Tris(nitrato-O)nitrosylruthenium

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Sept - 30 Sept 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tris(nitrato-O)nitrosylruthenium

EC Number:

252-068-8

EC Name:

Tris(nitrato-O)nitrosylruthenium

Cas Number:

34513-98-9

Molecular formula:

N4O10Ru

IUPAC Name:

Tris(nitrato-O)nitrosylruthenium

Test material form:

not specified

Details on test material:

- Name of test material (as cited in study report): Tris(nitrato-O)nitrosylruthenium
- Physical state: solid
- Analytical purity:
- Composition of test material, percentage of components: 28.84% ruthenium
- Date recieved: 09 December 2010
- Expiry date: 09 December 2011
- Storage conditions: room temperature and in the dark

Specific details on test material used for the study:

Tris(nitrato-O)nitrosylruthenium
purity: 31.8% Ru

Method

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:

Mammalian liver post-mitochondrial fraction (S-9)
S-9 prepared from male Sprague Dawley rats induced with Aroclor 1254.
S-9 supplied as lyophilized S-9 mix (MutazymeTM), stored frozen at <-10°C, and thawed and reconstituted with purified water to provide a 10% S-9 mix just prior to use.
Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P-450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities).
Treatments were carried out both in the absence and presence of S-9 by addition of either buffer solution or 10% S-9 mix respectively.

Test concentrations with justification for top dose:

Treatments in this study were performed using solutions of test item in vehicle up to a maximum concentration of 5000 μg/plate in Experiment 1, in order that initial treatments were performed up to this maximum recommended concentration according to current regulatory guidelines (OECD, 1997).
For Experiment 2 the maximum concentration tested was selected on the basis of toxicity seen in Experiment 1.
Toxicity assessed as diminution of background bacterial lawn and/or marke d reduciton in revertant numbers.
Experiment 1: 5, 16, 50, 160, 500, 1600, 5000 µg/plate (+ and - S9)
Experiment 2: 250, 500, 750, 1500, 2000, 3500, 5000 µg/plate (+ and - S9), treatments +S9 further modified by inclusion of pre-incubation step.

Vehicle / solvent:

Preliminary solubility data indicated that Tris(nitrato-O)nitrosylruthenium was
soluble in dimethylformamide (DMF) at concentrations equivalent to at least
58.3 mg/mL.
Test article stock solutions were prepared by formulating Tris(nitrato-
O)nitrosylruthenium under subdued lighting in DMF with the aid of vortex mixing
and warming at 37°C (as required), to give the maximum required treatment
concentration. Subsequent dilutions were made using DMF. The test article solutions
were protected from light and used within approximately 3.5 hours of initial
formulation.

Controls

Negative solvent / vehicle controls:

yes

Remarks:

0.1 mL DMF

Positive controls:

yes

Remarks:

0.05 mL additions

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene

Details on test system and experimental conditions:

0.1 mL volume additions of test article suspension were used for all treatments.
Plating details:
-0.1 mL of bacterial culture
-0.1 mL of test article suspension/vehicle control or 0.05 mL of positive control
-0.5 mL of 10% S-9 mix or buffer solution,
followed by rapid mixing and pouring on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated protected from light for 3 days in an incubator set to 37°C. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted.
As the results of Experiment 1 in the presence of S-9 were equivocal, treatments in
the presence of S-9 in Experiment 2 included a pre-incubation step. Quantities of test
article, vehicle control solution or positive control, bacteria and S-9 mix detailed
above, plus an additional 0.5 mL of 100 mM sodium phosphate buffer (pH 7.4) were
mixed together and placed in an orbital incubator set to 37°C for 20 minutes, before
the addition of 2 mL of supplemented molten agar at 45±1°C. Plating of these
treatments then proceeded as for the normal plate-incorporation procedure. In this
way it was hoped to increase the range of mutagenic chemicals that could be detected
in the assay.
The addition of 0.5 mL of 100 mM sodium phosphate buffer (pH 7.4) to these
Experiment 2 treatments in the presence of S-9 was employed as DMF, and some
other organic solvents, are known to be near to toxic levels when added at volumes of
0.1 mL in this assay system when employing the pre-incubation methodology. By
employing the modification indicated, the DMF concentration in the pre-incubation
mix was decreased, and it was hoped that this would minimise or eliminate any toxic
effects of the solvent that may have otherwise occurred. In order to ‘correct’ for the
additional volume in the pre-incubation mix, these were plated out using 2 mL of
1.125% supplemented soft agar, therefore the additions to each plate were comparable
to that of the plate incorporation treatments.

Rationale for test conditions:

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 or TA100) or ≥3-fold (in strains TA1535 or 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 none of the above criteria were met.

Statistics:

triplicate plates per concentration.
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

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

>1.5-fold increase, reproducible across 2 independent experments

Cytotoxicity / choice of top concentrations:

cytotoxicity

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 98

Metabolic activation:

without

Genotoxicity:

ambiguous

Remarks:

<2-fold increase, but some evidence of concentration-response relationship and reproducible amongst 2 experiments

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In each experiment the change in pH units across the concentration range was >1,
which the Sponsor considered was related to the acidity of the test item. As the assay
system is buffered, and the bacteria are tolerant of low pH values, treatments
proceeded as planned.

It may be noted that one vehicle control replicate plate count from the Experiment 1
treatments of strain TA102 in the absence of S-9 was invalidated as uncharacteristic
and unrepresentative of these strain treatments, as the plate count was significantly
below the historical control range, and the data are not reported. Sufficient data points
remained for these strain data to be accepted as valid.

Following Tris(nitrato-O)nitrosylruthenium treatments of strain TA102 in the absence
and presence of S-9, concentration-related (up to the lower limit of toxicity) increases
in revertant numbers that achieved or exceeded 1.5-fold the vehicle control level were
observed. These increases were
reproducible across the two independent experiments, and therefore were sufficient to
be considered as clear evidence of Tris(nitrato-O)nitrosylruthenium mutagenic
activity in this strain in this assay system.
No clear and concentration-related increases in revertant numbers were observed
following Tris(nitrato-O)nitrosylruthenium treatments in the absence and presence of
S-9 in any other tester strains, although small increases in revertant numbers were
observed in strain TA98 in the absence of S-9. These
increases provided at least some evidence of a concentration-relationship and were
reproducible across the two experiments, but failed to achieve 2-fold the concurrent
vehicle control level. These increases were considered as possible further evidence of
the Tris(nitrato-O)nitrosylruthenium mutagenic activity described in strain TA102.

Remarks on result:

mutagenic potential (based on QSAR/QSPR prediction)

Any other information on results incl. tables

Following Tris(nitrato-O)nitrosylruthenium treatments of strain TA102 in the absence

and presence of S-9, concentration-related (up to the lower limit of toxicity) increases

in revertant numbers that achieved or exceeded 1.5-fold the vehicle control level were

observed. These increases were reproducible across the two independent experiments,

and therefore were sufficient to be considered as clear evidence of Tris(nitrato-

O)nitrosylruthenium mutagenic activity in this strain in this assay system.

No clear and concentration-related increases in revertant numbers were observed

following Tris(nitrato-O)nitrosylruthenium treatments in the absence and presence of

S-9 in any other tester strains, although small increases in revertant numbers were

observed in strain TA98 in the absence of S-9. These increases provided at least some

evidence of a concentration-relationship and were reproducible across the

two experiments, but failed to achieve 2-fold the concurrent vehicle control level.

These increases were considered as possible further evidence of the Tris(nitrato-

O)nitrosylruthenium mutagenic activity described in strain TA102.

Applicant's summary and conclusion

Conclusions:

It was concluded that Tris(nitrato-O)nitrosylruthenium induced mutation in histidinerequiring
strain TA102 of Salmonella typhimurium in the absence and presence of
metabolic activation, when tested under the conditions of this study. These conditions
included treatments at concentrations up to 5000 μg/plate (the maximum
recommended concentration according to current regulatory guidelines), in the
absence and in the presence of a rat liver metabolic activation system (S-9). Small
increases in revertant numbers observed following Tris(nitrato-O)nitrosylruthenium
treatments of strain TA98 in the absence of S-9 may have been further evidence of
mutagenic activity.

Executive summary:

Tris(nitrato-O)nitrosylruthenium 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 Tris(nitrato-O)nitrosylruthenium treatments in this study were performed using

formulations prepared in dimethylformamide (DMF).

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 Tris(nitrato-

O)nitrosylruthenium at 5, 16, 50, 160, 500, 1600 and 5000 μg/plate. Following these

treatments, evidence of toxicity was observed at 5000 μg/plate in all strains but in the

absence of S-9 only.

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 250-5000 μg/plate, in order to examine more closely those concentrations of

Tris(nitrato-O)nitrosylruthenium approaching the maximum test concentration and

considered therefore most likely to provide evidence of any mutagenic activity. In

addition, all treatments in the presence of S-9 were further modified by the inclusion

of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic

chemicals that could be detected using this assay system. Following these treatments,

evidence of toxicity was observed in all strains in the absence and presence of S-9,

and extending down to either 2000, 3500 or 5000 μg/plate in each case.

The test article was completely soluble in the aqueous assay system at all

concentrations treated, in each of the experiments performed.

Vehicle and positive control treatments were included for all strains in both

experiments. The mean numbers of revertant colonies were comparable with

acceptable ranges for vehicle control treatments, and were elevated by positive control

treatments.

Following Tris(nitrato-O)nitrosylruthenium treatments of strain TA102 in the absence

and presence of S-9, concentration-related (up to the lower limit of toxicity) increases

in revertant numbers that achieved or exceeded 1.5-fold the vehicle control level were

observed. These increases were reproducible across the two independent experiments,

and therefore were sufficient to be considered as clear evidence of Tris(nitrato-

O)nitrosylruthenium mutagenic activity in this strain in this assay system.

No clear and concentration-related increases in revertant numbers were observed

following Tris(nitrato-O)nitrosylruthenium treatments in the absence and presence of

S-9 in any other tester strains, although small increases in revertant numbers were

observed in strain TA98 in the absence of S-9. These increases provided at least some

evidence of a concentration-relationship and were reproducible across the

two experiments, but failed to achieve 2-fold the concurrent vehicle control level.

These increases were considered as possible further evidence of the Tris(nitrato-

O)nitrosylruthenium mutagenic activity described in strain TA102.

It was concluded that Tris(nitrato-O)nitrosylruthenium induced mutation in histidinerequiring

strain TA102 of Salmonella typhimurium in the absence and presence of

metabolic activation, when tested under the conditions of this study. These conditions

included treatments at concentrations up to 5000 μg/plate (the maximum

recommended concentration according to current regulatory guidelines), in the

absence and in the presence of a rat liver metabolic activation system (S-9). Small

increases in revertant numbers observed following Tris(nitrato-O)nitrosylruthenium

treatments of strain TA98 in the absence of S-9 may have been further evidence of

mutagenic activity.