Tris(nitrato-O)nitrosylruthenium

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genetic toxicity potential of tris(nitrato-O)nitrosyl ruthenium is determined using an in vitro reverse mutation assay with bacteria according to protocol OECD 471 (GLP compliant) and an in vitro micronucleus assay, using duplicate human lymphocyte cultures prepared from the pooled blood of two female donors, in a single experiment according to protocol OECD487 (GLP compliant). Both in vitro assays provided evidence of tris(nitrato-O)nitrosyl ruthenium mutagenic activity.

A repeat investigative AMES assay with strains Salmonella strain TA102 and E. coli strain WP2 uvrA was performed. In absence of any indication of mutagenic activity in the E. coli strain WP2 uvrA, when tested under the same treatment conditions, is considered to support the supposition that reactive oxygen species could be responsible for the Tris(nitrato-O)nitrosylruthenium mutagenic activity in strain TA102, as strain WP2 uvrA is considered to be less susceptible to the mutagenic effects of reactive oxygen species.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

24 Nov - 21 Dec 2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This is an investigative follow on study from the earlier AMES (Ballantyne, 2020) and in vitro HLM (Chirom, 2020) studies, where positive results were obtained, which may be linked to reactive oxygen species. In the previous Ames study (Ballantyne 2020), a mutagenic response was only evident in Salmonella strain TA102, and therefore in this study testing was conducted in this strain, and also in E. coli strain WP2 uvrA. This E. coli strain is a regulatory acceptable (OECD 471) alternative tester strain to Salmonella TA102, but it is considered less susceptible to the mutagenic effects of reactive oxygen species. By assessing whether a clear mutagenic response was observed in one or both strains, assessment could be made on whether reactive oxygen species were likely to be responsible for the observed mutagenic activity.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

corrected in 2020

Deviations:

yes

Remarks:

This is an investigative study, and due to the abridged strains tested in this study (cfr justification for strain selection), it does not meet with the formal requirements of the standard regulatory guideline (OECD Guideline 471)

Principles of method if other than guideline:

although the study does not meet the formal OECD471 guidelines due to the strain selection, the applied experimental protocol is in line with that of OECD471.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

31.8%
solution in nitric acid

Target gene:

mutant gene histidine (S. typhimurium) and tryptophan (E. coli)

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation system:

The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it was prepared from male Sprague Dawley rats induced with β-Naphthoflavone/Phenobarbital. The S-9 was 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:

Final concentration (µg test item / plate) for mutation experiments 1 & 2: 250, 500, 1000, 1500, 2000, 3000, 4000, 5000

5000 µg/plate is the maximum recommended concentration according to current regulatory guidelines (OECD, 1997, as corrected in 2020), and is used for experiments 1 and 2.

Vehicle / solvent:

Tris(nitrato-O)nitrosylruthenium was soluble in dimethylformamide (DMF) at concentrations up to at least 310.17 mg/mL (cfr. Ballantyne 2020).
Test article stock solutions were prepared by formulating Tris(nitrato-O)nitrosylruthenium under subdued lighting in DMF, with the aid of vortex mixing (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 2 hours of initial formulation.
0.025 mL volume additions were used for all treatments.

Negative solvent / vehicle controls:

yes

Remarks:

DMF, 0.025 mL additions per plate as the test article treatments

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

mitomycin C

other: 2-aminoanthracene

Remarks:

Positive controls comprised treatments with the appropriate stock positive control solution using 0.05 mL additions.
Mitomycin C: 0.2 µg/plate (TA102)
4-nitroquinoline 1-oxide: 2 µg/plate (WP2 uvrA)
2-aminoanthracene:15 (WP2 uvrA) and 20 µg/plate (TA102)

Details on test system and experimental conditions:

As the study is intended to assess whether active oxygen species may be responsible for any observed mutagenic activity, a pre-incubation methodology was employed, as this is considered more sensitive in the detection of mutagenicity due to reactive oxygen species.

L-histidine HCl (in 250 mM MgCl2) or DL-tryptophan (for S. typhimurium or E. coli strain respectively) together with D-biotin (for S. typhimurium strains) were added at the time of plating, by supplementing the top agar.

Bacteria:
One strain of Salmonella typhimurium bacteria (TA102) and one strain of Escherichia coli bacteria (WP2 uvrA) were used in this study. Strain TA102 was derived from cultures originally obtained from Covance Laboratories Inc., USA. Strain WP2 uvrA was obtained from the Cancer Research Unit, University of York, UK. For all assays, bacteria were cultured in an incubator set to 37°C for 10 hours in nutrient broth, containing ampicillin and tetracycline for strain TA02 only, to provide bacterial cultures in the range of approximately 108 to 109 cells/mL, confirmed by cell density assessments for each culture. Incubation was carried out with shaking in an anhydric incubator, set to turn on using a timer switch. All treatments were completed within 6 hours of the end of the incubation period.
The inocula were taken from master plates or vials of frozen cultures, which had been checked for strain characteristics (histidine or tryptophan dependence, rfa character and uvrB character if applicable, and resistance to ampicillin or ampicillin plus tetracycline).

4.2 Mutation Experiments
Tris(nitrato-O)nitrosylruthenium was tested for mutation (and toxicity) in one strain of Salmonella typhimurium (TA102) and one strain of Escherichia coli (WP2 uvrA) in two separate experiments at the concentrations detailed previously, using triplicate plates without and with S-9 for test article, vehicle and positive controls. These platings were achieved by the following sequence of additions to sterile pre-incubation tubes:
• 0.1 mL bacterial culture
• 0.025 mL of test article solution or vehicle control or 0.05 mL of positive control
• 0.5 mL 10% S-9 mix or buffer solution.
The contents of each tube were mixed together and placed in an orbital incubator set to 37C for 20 minutes, before the addition of 2 mL (0.9%) molten agar at 45±1°C, 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 2 to 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.
To address the extensive toxicity observed following Experiment 1, all the treatments in Experiment 2 were performed with the addition of 0.5 mL of 100 mM sodium phosphate buffer (pH 7.4). 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, so that when plated, the top agar concentration was the same as for Experiment 1.

Toxicity Assessment
The background lawns of the plates were examined for signs of toxicity. Revertant plate count data were also assessed, as a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response was also considered as evidence of toxicity. Where mutation data from fewer than five treatment concentrations was obtained, an evaluation of the mutation data for the study as a whole was made.

Colony Enumeration
Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually where confounding factors such as bubbles or splits in the agar affected the accuracy of the automated counter.

Evaluation criteria:

The mutagenicity of the test article was assessed in each strain separately.
For valid data, the test article was considered to be mutagenic in strain TA102 if:
1. A concentration related increase in revertant numbers was ≥1.5-fold the concurrent vehicle control value
2. Any observed response was reproducible under the same treatment conditions.
For valid data, the test article was considered to be mutagenic in strain WP2 uvrA if:
1. A concentration related increase in revertant numbers was ≥2-fold the concurrent vehicle control value
2. Any observed response was reproducible under the same treatment conditions.
The test article was considered positive in a test strain in this assay if both of the above criteria for that strain were met.
The test article was considered negative in a test strain in this assay if the above criteria for that strain were not 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.
The mutation assessment of the test article in the two tester strains was compared. Where the test article was clearly mutagenic in strain TA102 but not clearly mutagenic in strain WP2 uvrA, this was considered to support the mode of action that a reactive oxygen species may be responsible for the observed mutagenic activity.

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.

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

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

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

positive

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:

Toxicity, Solubility and Concentration Selection
Mutation Experiment 1 treatments of both the tester strains were performed in the absence and in the presence of S-9, using final concentrations of Tris(nitrato-O)nitrosylruthenium at 250, 500, 1000, 1500, 2000, 3000, 4000 and 5000 µg/plate. Following these Experiment 1 treatments, evidence of toxicity was observed on all plates treated at 1500 µg/plate and above, and was manifest as a thinning of the background bacterial lawn, with or without a marked reduction in revertant numbers, or a complete killing of the test bacteria. This toxicity was more extensive than previously observed, which was attributed to a combination of the use of a pre-incubation methodology (which often provides more toxicity than with corresponding plate incorporation methodology treatments), together with the vehicle (DMF) used in this study, which can cause vehicle–related toxicity when used with the pre-incubation methodology in this assay system.
For the Mutation Experiment 2 treatments of both the tester strains, a pre-incubation methodology was again employed in the absence and in the presence of S-9, but an additional 0.5 mL of 100 mM Sodium phosphate buffer (pH 7.4) was added to each pre-incubation mix, in an attempt to reduce any toxic effects of the vehicle or the test article treatments. Treatments were performed using the same treatment concentrations used for Mutation Experiment 1, but with an additional lower treatment concentration (125 µg/plate) also included. Following these treatments, evidence of toxicity was again observed, but was limited to treatments at 3000 µg/plate and above in both strains in the absence and presence of S-9.
The test article was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed.

Mutation
Following Tris(nitrato-O)nitrosylruthenium treatments of both the tester strains in the absence and presence of S-9, notable increases in revertant numbers were only observed in strain TA102 (in both the absence and presence of S-9). These increases were concentration-related up to the lower limit of toxicity, but only exceeded 1.5 fold the concurrent vehicle control level in Mutation Experiment 1 in the absence of S-9 (see Table 8.1). Increases in revertant numbers that approached, but did not quite achieve 1.5-fold the vehicle control level, were observed in strain TA102 treatments in the absence and presence of S-9 in Experiment 2 (see Table 8.3 and Table 8.4), but no notable increase was observed in this strain in the presence of S-9 in Experiment 1. The increase in strain TA102 in the absence of S-9 was therefore considered to be reproducible, although close to the limit of detection in this assay system, and was considered indicative of a relatively weak Tris(nitrato-O)nitrosylruthenium mutagenic response.
No notable increases were observed in strain WP2 uvrA in either experiment in either the absence or presence of S-9, and therefore this study has provided no indication of any Tris(nitrato-O)nitrosylruthenium mutagenic activity in this strain.
As a mutagenic response (albeit relatively weak) was observed in strain TA102 but not in strain WP2 uvrA, this is consistent with reactive oxygen species being responsible for the observed Tris(nitrato-O)nitrosylruthenium mutagenic activity.

Conclusions:

It was concluded that this study provided evidence of relatively weak mutagenic activity in strain TA102 in the absence of a rat liver metabolic activation system (S- 9), when Tris(nitrato-O)nitrosylruthenium was tested under the treatment conditions employed for this study. These conditions included treatments up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines, and in this study a toxic concentration), in the absence and in the presence of S-9. The absence of any indication of mutagenic activity in the E. coli strain WP2 uvrA, when tested under the same treatment conditions in this study, is considered to support the supposition that reactive oxygen species could be responsible for the Tris(nitrato-O)nitrosylruthenium mutagenic activity in strain TA102, as strain WP2 uvrA is considered to be less susceptible to the mutagenic effects of reactive oxygen species.

Executive summary:

Tris(nitrato-O)nitrosylruthenium was assayed for mutation in one histidine-requiring strain (TA102) of Salmonella typhimurium, and one tryptophan-requiring strain (WP2 uvrA) of Escherichia coli, both in the absence and in the presence of metabolic activation by a β-Naphthoflavone/Phenobarbital-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments.

This is an investigative follow on study from an earlier AMES (Ballantyne, 2020) and in vitro HLM (Chirom, 2020) assay, where positive results were obtained, which may be linked to reactive oxygen species. In the previous Ames study (Ballantyne 2020), a mutagenic response was only evident in Salmonella strain TA102, and therefore in this study testing was conducted in this strain, and also in E. coli strain WP2 uvrA. This E. coli strain is a regulatory acceptable (OECD 471) alternative tester strain to Salmonella TA102, but it is considered less susceptible to the mutagenic effects of reactive oxygen species. By assessing whether a clear mutagenic response was observed in one or both strains, assessment could be made on whether reactive oxygen species were likely to be responsible for the observed mutagenic activity.

All Tris(nitrato-O)nitrosylruthenium treatments in this study were performed using formulations prepared in dimethylformamide (DMF). As the study was intended to assess whether active oxygen species may be responsible for any observed mutagenic activity, a pre-incubation methodology was employed, as this is considered more sensitive in the detection of mutagenicity due to reactive oxygen species.

Mutation Experiment 1 treatments of both the tester strains were performed in the absence and in the presence of S-9, using final concentrations of Tris(nitrato-O)nitrosylruthenium at 250, 500, 1000, 1500, 2000, 3000, 4000 and 5000 µg/plate. The treatment concentrations were selected to investigate more closely the concentration range over which increases in revertant numbers were previously observed. Following these Experiment 1 treatments, evidence of toxicity was observed on all plates treated at 1500 µg/plate and above. This toxicity was more extensive than previously observed, and this was attributed to the use of a pre-incubation methodology in this study (which often provides more toxicity than with corresponding plate incorporation methodology treatments), together with the vehicle (DMF) used in this study, which can cause vehicle–related toxicity when used with the pre-incubation methodology in this assay system.

For the Mutation Experiment 2 treatments of both the tester strains, a pre-incubation methodology was again employed in the absence and in the presence of S-9, but an additional 0.5 mL of 100 mM Sodium phosphate buffer (pH 7.4) was added to each pre-incubation mix, in an attempt to reduce any toxic effects of the vehicle or the test article treatments. Treatments were performed using the same treatment concentrations used for Mutation Experiment 1, but with an additional lower treatment concentration (125 µg/plate) also included. Following these treatments, evidence of toxicity was again observed, but was limited to treatments at 3000 µg/plate and above in both strains in the absence and presence of S-9.

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 both strains in each experiment. The 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 both the tester strains in the absence and presence of S-9, notable increases in revertant numbers were observed in strain TA102 in both the absence and presence of S-9. These increases were concentration-related up to the lower limit of toxicity, but only exceeded 1.5-fold the concurrent vehicle control level in Mutation Experiment 1 in the absence of S-9. Increases in revertant numbers that approached, but did not quite achieve 1.5-fold the vehicle control level, were observed in strain TA102 treatments in the absence and presence of S-9 in Experiment 2, but no notable increase was observed in this strain in the presence of S-9 in Experiment 1. The increase in strain TA102 in the absence of S-9 was therefore considered to be reproducible, although close to the limit of detection in this assay system, and was considered indicative of a relatively weak Tris(nitrato-O)nitrosylruthenium mutagenic response. No notable increases were observed in strain WP2 uvrA in either experiment in either the absence or presence of S-9, and therefore this study has provided no indication of any Tris(nitrato-O)nitrosylruthenium mutagenic activity in this strain.

As a mutagenic response (albeit relatively weak) was only observed in strain TA102 and not in strain WP2 uvrA, this is consistent with the supposition that reactive oxygen species are responsible for the observed Tris(nitrato-O)nitrosylruthenium mutagenic activity.

It was concluded that this study provided evidence of relatively weak Tris(nitrato-O)nitrosylruthenium mutagenic activity in strain TA102 in the absence of a rat liver metabolic activation system (S-9), when tested under the treatment conditions employed for this study. These conditions included treatments up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines, and in this study a toxic concentration), in the absence and in the presence of S-9. The absence of any indication of mutagenic activity in the E. coli strain WP2 uvrA, when tested under the same treatment conditions in this study, is considered to support the supposition that reactive oxygen species could be responsible for the Tris(nitrato-O)nitrosylruthenium mutagenic activity in strain TA102, as strain WP2 uvrA is considered to be less susceptible to the mutagenic effects of reactive oxygen species.

Reference 2

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

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

Specific details on test material used for the study:

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

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.

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.

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

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)

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.

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.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Oct 2019 - 14 Jan 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

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

Metabolic activation:

with and without

Metabolic activation system:

The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation
was obtained from Molecular Toxicology Incorporated, USA where it was prepared
from male Sprague Dawley rats induced with Aroclor 1254. The S-9 was 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 150 mM KCl or 10% S-9 mix respectively. The final S-9 volume in the test
system was 1% (v/v).

Test concentrations with justification for top dose:

maximum conc of 2000 μg/mL was
selected for the cytotoxicity Range-Finder Experiment (RFE), in order that treatments were
performed up to the maximum recommended conc according to current
regulatory guidelines. Concentrations for the MN
Experiment were selected based on the results of this cytotoxicity RFE.

Slides from the cytotoxicity RFE were examined, uncoded, for
proportions of mono-, bi- and multinucleate cells, to a minimum of 200 cells per
conc. From these data the replication index (RI) was determined.
RI, which indicates the relative number of nuclei compared to vehicle controls, was
determined. Relative RI (expressed in terms of percentage) for each treated culture was calculated.
Cytotoxicity (%) is expressed as (100 – Relative RI).
Cytotoxicity was assessed from enough treatment concentrations to determine
whether chemically induced cell cycle delay had occurred.
A suitable range of concentrations was selected for the MN Experiment
based on these toxicity data.
Slides were examined, uncoded, for RI to a minimum of 500 cells per culture to
determine whether chemically induced cell cycle delay or toxicity had occurred.
The highest conc selected for MN analysis following 3+21 hour
treatment in the absence and presence of S-9 was the highest concentration tested
(2000 μg/mL).
The highest conc selected for MN analysis following 24+24 hour
treatment in the absence of S-9 was one at which 50-60% cytotoxicity was achieved.
Slides from the highest selected conc and two lower conc were
taken for microscopic analysis, such that a range of cytotoxicity from maximum to
little was covered.

The following conc ranges were tested:
RFE (3+21h,-S9; 3+21h,+S9; 24+24h,-S9): 0.7256 to 200 mg/mL conc range, 7.256 to 2000 µg/ml final conc range
MN experiment (3+21h,-S9; 3+21h,+S9): 25 to 200 mg/mL conc range, 250 to 2000 µg/mL final conc range
MN experiment (24+24h,-S9): 1 to 150 mg/mL conc range, 10 to 1500 µg/mL final conc range

Vehicle / solvent:

Preliminary solubility data indicated that Tris(nitrato-O)nitrosylruthenium was
soluble in dimethylformamide (DMF) at concentrations up to at least 310.17 mg/mL.
The solubility limit in culture medium was in excess of 3101.7 μg/mL as indicated by
a lack of any visible precipitation at this concentration over a period of approximately
24 hours after test article addition. A maximum concentration of 2000 μg/mL was
selected for the cytotoxicity Range-Finder Experiment, in order that treatments were
performed up to the maximum recommended concentration according to current
regulatory guidelines.
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.

Untreated negative controls:

yes

Remarks:

Cultures treated with culture medium alone

Negative solvent / vehicle controls:

yes

Remarks:

sterile DMF

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

vinblastine

Remarks:

In the Micronucleus Experiment, CPA was dissolved in anhydrous analytical grade dimethyl sulphoxide (DMSO), frozen (<-50ºC), thawed immediately prior to use and diluted accordingly. VIN and MMC were dissolved in purified water immediately prior to use.

Details on test system and experimental conditions:

pH measurements of the stock formulation and its subsequent dilutions in the primary
vehicle were taken prior to treatment in the Micronucleus Experiment. A gradual
decrease in pH units with increase in concentration was noted. The change
in pH units across the concentration range was observed to be >1. As the test system
possesses buffering capacity, treatment was proceeded as planned. Following the
treatment period, pH measurements in post treatment medium samples were also
taken for all concentrations tested in the Micronucleus Experiment. No marked
changes in pH (shifts of greater than 1 pH unit) were observed, as compared to the
concurrent vehicle.

Blood from two healthy, non-smoking female volunteers from a panel of donors at
Covance was used for each experiment.
No donor was suspected of any virus infection or exposed to high levels of radiation
or hazardous chemicals. All donors are non-smokers and are not heavy drinkers of
alcohol. Donors were not taking any form of medication (contraceptive pill excluded).
The measured cell cycle time of the donors used at Covance, Harrogate falls within
the range 13±2 hours. For each experiment, an appropriate volume of whole blood
was drawn from the peripheral circulation into heparinised tubes within one day of
culture initiation. Blood was stored refrigerated and pooled using equal volumes from
each donor prior to use.
Whole blood cultures were established in sterile disposable centrifuge tubes by
placing 0.4 mL (Range-Finder experiment) and 0.6 mL (Micronucleus Experiment) of
pooled heparinised blood into 8.5 mL (Range-Finder Experiment) and 8.3 mL
(Micronucleus Experiment) pre-warmed (in an incubator set to 37°C) HEPESbuffered
RPMI medium containing 10% (v/v) heat inactivated foetal calf serum and
0.52% penicillin / streptomycin, so that the final volume following addition of S-9
mix/KCl and the test article in its chosen vehicle was 10 mL. The mitogen
Phytohaemagglutinin (PHA, reagent grade) was included in the culture medium at a
concentration of approximately 2% of culture to stimulate the lymphocytes to divide.
Blood cultures were placed in an incubator set to 37°C for approximately 48 hours
and rocked continuously.

Micronucleus experiment:
S-9 mix or KCl (1 mL per culture) was added appropriately. Cultures were treated
with the test article, vehicle, culture medium for the UTC or positive controls (0.1 mL
per culture).
The final culture volume was 10 mL. Cultures were placed in an incubator set to 37°C
for the designated exposure time.
For removal of the test article, cells were pelleted (approximately 300 g, 10 minutes),
washed twice with sterile saline (pre-warmed in an incubator set to 37°C), and
resuspended in fresh pre-warmed medium containing foetal calf serum and penicillin /
streptomycin. Cyto-B (formulated in DMSO) was added to post wash-off culture
medium to give a final concentration of 6 μg/mL per culture.
At the defined sampling time, cultures were centrifuged at approximately 300 g for
10 minutes, the supernatant removed and discarded and cells resuspended in 4 mL
(hypotonic) 0.075 M KCl at approximately 37°C for 4 minutes to allow cell swelling
to occur. Cells were then fixed by dropping the KCl suspension into fresh, cold
methanol/glacial acetic acid (7:1, v/v). The fixative was changed by centrifugation
(approximately 300 g, 10 minutes) and resuspension. This procedure was repeated as
necessary (centrifuging at approximately 1250 g, 2-3 minutes) until the cell pellets
were clean.
Lymphocytes were kept in fixative at 2-8°C prior to slide preparation for a minimum
of 3 hours to ensure that cells were adequately fixed. Cells were centrifuged
(approximately 1250 g, 2-3 minutes) and resuspended in a minimal amount of fresh
fixative (if required) to give a milky suspension. Several drops of cell suspension
were gently spread onto multiple clean, dry microscope slides. Slides were air-dried
prior to staining. Slides were stained by immersion in 12.5 μg/mL Acridine Orange in
phosphate buffered saline (PBS), pH 6.8 for approximately 10 minutes and washed
with PBS (with agitation) for a few seconds. The quality of the staining was checked.
Slides were air-dried and protected from light at room temperature. Immediately prior
to analysis 1-2 drops of PBS were added to the slides before mounting with glass
coverslips.

Micronucleus Analysis
Scoring was carried out using fluorescence microscopy.
Binucleate cells were only included in the analysis if all of the following criteria were met:
1. The cytoplasm remained essentially intact, and
2. The daughter nuclei were of approximately equal size.
A micronucleus was only recorded if it met the following criteria:
1. The micronucleus had the same staining characteristics and a similar morphology
to the main nuclei, and
2. Any micronucleus present was separate in the cytoplasm or only just touching a
main nucleus, and3. Micronuclei were smooth edged and smaller than approximately one third the
diameter of the main nuclei.
For each treatment regime, two vehicle control cultures were analysed for
micronuclei. As the vehicle control data were considered acceptable, UTC were not
analysed.
Slides from the positive control treatments were checked to ensure that the system
was operating satisfactorily. One concentration from each positive control, which
gave satisfactory responses in terms of quality and quantity of binucleated cells and
numbers of micronuclei, was analysed. This pre-analysis slide check was conducted
under non-blinded conditions.
All slides for analysis were coded by an individual not connected with the scoring of
the slides, such that analysis was conducted under blind conditions. Labels with only
the study number, assay type, experiment number, the sex of the donor and the code
were used to cover treatment details on the slides.
One thousand binucleate cells from each culture (2000 per concentration) were
analysed for micronuclei. The number of cells containing micronuclei on each slide
was recorded.
Nucleoplasmic bridges (NPBs) between nuclei in binucleate cells were recorded
during micronucleus analysis to provide an indication of chromosome rearrangement.
Various mechanisms may lead to NPB formation following DNA misrepair of strand
breaks in DNA (Thomas et al., 2003). In this assay, binucleate cells with NPBs were
recorded as part of the micronucleus analysis.

Evaluation criteria:

For valid data, the test article was considered to induce clastogenic and/or aneugenic
events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more
concentrations was observed
2. An incidence of MNBN cells at such a concentration that exceeded the normal
range in both replicates was observed
3. A concentration-related increase in the proportion of MNBN cells was observed
(positive trend test).
The test article was considered positive in this assay if all of the above criteria were
met.
The test article was considered negative in this assay if none of the above criteria
were met.
Following FISH analysis:
1. The test article was considered aneugenic if it induced predominantly
centromere-positive micronuclei
2. The test article was considered clastogenic if it induced predominantly
centromere-negative micronuclei.
To conclude whether the test article was acting via a predominantly clastogenic or
aneugenic mechanism, the test article data were analysed in conjunction with the
reference aneugenic and clastogenic responses.
Results which only partially satisfied the above criteria were dealt with on a case-bycase
basis. Evidence of a concentration-related effect was considered useful but not
essential in the evaluation of a positive result (Scott et al., 1990). Biological relevance
was taken into account, for example consistency of response within and between
concentrations, or effects occurring only at very toxic concentrations (Thybaud et al.,
2007).

Statistics:

After completion of scoring and decoding of slides, the numbers of binucleate cells
with micronuclei (MNBN cells) in each culture were obtained.
The proportions of MNBN cells in each replicate were used to establish acceptable
heterogeneity between replicates by means of a binomial dispersion test (Richardson
et al., 1989).
The proportions of MNBN cells for each treatment condition were compared with the
proportion in vehicle controls by using Fisher's exact test (Richardson et al., 1989). A
Cochran-Armitage trend test was applied to each treatment condition. Probability
values of p≤0.05 were accepted as significant.

Acceptance criteria:
The assay was considered valid if the following criteria were met:
1. The binomial dispersion test demonstrated acceptable heterogeneity (in terms of
MNBN cell frequency) between replicate cultures, particularly where no positive
responses were seen
2. The frequency of MNBN cells in vehicle controls fell within the historical vehicle
control (normal) ranges
3. The positive control chemicals induced statistically significant increases in the
proportion of cells with micronuclei. Both replicate cultures at the positive control
concentration analysed under each treatment condition demonstrated MNBN cell
frequencies that clearly exceeded the current historical vehicle control ranges
4. A minimum of 50% of cells had gone through at least one cell division (as
measured by binucleate + multinucleate cell counts) in vehicle control cultures at
the time of harvest
5. The maximum concentration analysed under each treatment condition met the
criteria specified in Section 4.7.
The FISH analysis was considered valid if the following criteria were met:
1. The micronuclei in cultures treated with the clastogen (MMC) were
predominantly centromere-negative
2. The micronuclei in cultures treated with the aneugen (VIN) were predominantly
centromere-positive.

Key result

Species / strain:

other: cultured human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Treatment of cells with Tris(nitrato-O)nitrosylruthenium for 3+21 hours and
24+24 hours in the absence of S-9, resulted in frequencies of MNBN cells that were
significantly higher (p≤0.01 or p≤0.001), compared to those observed in concurrent
vehicle control cultures for all concentrations analysed for both treatment conditions. With the exception of a single
replicate at the lowest concentration (500 μg/mL) for the 3+21 hour treatment in the
absence of S-9 , the MNBN cell frequencies of all other test
article treated cultures (both treatment conditions) exceeded the normal ranges.
Statistically significant linear trends (p≤0.001) were observed
following both treatment conditions in the absence of S-9.
Treatment of cells for 3+21 hours in the presence of S-9 resulted in frequencies of
MNBN cells that were significantly higher (p≤0.05 or p≤0.01), compared to those
observed in concurrent vehicle control cultures, for the lowest and highest
concentrations analysed (500 and 2000 μg/mL), with a statistically significant linear
trend (p≤0.05). However, these increases were small in
magnitude such that the MNBN cell frequency of only a single culture at the highest
concentration analysed (2000 μg/mL, inducing 31% cytotoxicity) marginally
exceeded the normal range (1.30% in comparison to the normal range of 0 to 1.20%).
Overall these small statistical increases were considered to
present weak evidence of a test article related effect under this test condition.
No test article related increases in cells with NPBs were observed (data not reported).

For the positive control compounds, treatment of cells with MMC (0.30 μg/mL) for
3+21 hours in the absence of S-9 induced 90% centromere-negative micronuclei
(C- MN) and 10% centromere-positive micronuclei (C+ MN) from a total of 100 MN
analysed, indicating a predominantly clastogenic mechanism. Treatment with VIN
(0.04 μg/mL) for 24+24 hours in the absence of S-9 induced 48% C- MN and
52% C+ MN from a total of 101 MN analysed, indicating a slightly predominant
aneugenic mechanism, although the ratio was close to 50:50.
Treatment with Tris(nitrato-O)nitrosylruthenium for 3+21 hours in the absence of S-9
at 2000 μg/mL induced 84% C- MN and 16% C+ MN from a total of 98 MN
analysed.
Treatment with Tris(nitrato-O)nitrosylruthenium for 24+24 hours in the absence of
S-9 at 200, 400 and 700 μg/mL induced 96%, 96% and 98% C- MN (4%, 4% and 2%
C+ MN), respectively, from a total of 100 MN analysed at each concentration.
The FISH analysis data for the 3+21 hour and 24+24 hour treatments in the absence
of S-9 confirm a predominantly clastogenic (chromosome breakage) mechanism for
both treatment conditions.

Remarks on result:

mutagenic potential (based on QSAR/QSPR prediction)

Treatment	Concentration (µg/mL)	Cytotoxicity (%)^$	Mean MN Cell Frequency (%)	Historical Control Range (%)^#	Statistical Significance
3+21 -S9	Vehicle^a	-	0.40	0.00 to 1.01	-
	500.0	11	1.15		p≤0.01
	1000	23	1.85		p≤0.001
	2000	35	2.70		p≤0.001
	*MMC, 0.30	28	5.70		p≤0.001
3+21 +S9	Vehicle^a	-	0.45	0.10 to 1.20	-
	500.0	9	0.95		p≤0.05
	1000	18	0.85		NS
	2000	31	1.15		p≤0.01
	*CPA, 7.00	49	2.65		p≤0.001
24+24 -S9	Vehicle^a	-	0.35	0.10 to 0.80	-
	200.0	12	3.20		p≤0.001
	400.0	21	4.50		p≤0.001
	700.0	50	6.70		p≤0.001
	*VIN, 0.04	11	4.60		p≤0.001

^a             Vehicle control was DMF

*            Positive control

^#             95^thpercentile of the observed range

^$             Based on RI

MN        Micronucleated

NS         Not significant

Conclusions:

Tris(nitrato-O)nitrosylruthenium was tested in an in vitro micronucleus assay, using
duplicate human lymphocyte cultures prepared from the pooled blood of two female
donors, in a single experiment according to OECD487 (GLP compliant).
It is concluded that Tris(nitrato-O)nitrosylruthenium induced increases in the
frequency of micronuclei following treatments in cultured human peripheral blood
lymphocytes in the absence and presence of an Aroclor-induced rat liver metabolic
activation system (S-9). The maximum concentrations analysed for micronuclei were
either 2000 μg/mL (the maximum concentration recommended by the regulatory test
guidelines for the in vitro micronucleus assay) for the 3+21 hour treatments in the
absence or presence of S-9 or were limited by cytotoxicity for the 24+24 hour
treatments in the absence of S-9.
The use of FISH with pan-centromeric DNA probes demonstrated that for the
3+21 hour and 24+24 hour -S-9 treatments, micronuclei were generated via a
predominantly clastogenic mechanism (chromosome breakage) following treatment
with Tris(nitrato-O)nitrosylruthenium.

Executive summary:

Tris(nitrato-O)nitrosylruthenium was tested in an in vitro micronucleus assay, using

duplicate human lymphocyte cultures prepared from the pooled blood of two female

donors, in a single experiment. Treatments covering a broad range of concentrations,

separated by narrow intervals, were performed both in the absence and presence of

metabolic activation (S-9) from Aroclor 1254-induced rats. The test article was

formulated in dimethylformamide (DMF) and the highest concentrations tested in the

Micronucleus Experiment, were determined following a preliminary cytotoxicity

Range-Finder Experiment.

Treatments were conducted (as detailed in the following summary table) 48 hours

following mitogen stimulation by Phytohaemagglutinin (PHA). The test article

concentrations for micronucleus analysis were selected by evaluating the effect of

Tris(nitrato-O)nitrosylruthenium on the replication index (RI). Micronuclei were

analysed at three concentrations.

Appropriate negative (vehicle and untreated) control cultures were included in the test

system under each treatment condition. The proportion of micronucleated binucleate

(MNBN) cells in the vehicle cultures fell within the 95th percentile of the current

observed historical vehicle control (normal) ranges. It was therefore not considered

necessary to analyse the untreated control cultures. Mitomycin C (MMC) and

Vinblastine (VIN) were employed as clastogenic and aneugenic positive control

chemicals, respectively, in the absence of rat liver S-9. Cyclophosphamide (CPA) was

employed as a clastogenic positive control chemical in the presence of rat liver S-9.

Cells receiving these were sampled in the Micronucleus Experiment at 24 hours

(CPA, MMC) or 48 hours (VIN) after the start of treatment. All positive control

micronuclei.

All acceptance criteria were considered met and the study was therefore accepted as

valid.

Micronucleus Analysis

Treatment of cells with Tris(nitrato-O)nitrosylruthenium for 3+21 hours and

24+24 hours in the absence of S-9 resulted in frequencies of MNBN cells that were

significantly higher (p≤0.01 or p≤0.001), compared to those observed in concurrent

vehicle control cultures for all concentrations analysed for both treatment conditions.

With the exception of a single replicate at the lowest concentration (500 μg/mL) for

the 3+21 hour treatment in the absence of S-9, the MNBN cell frequencies of all other

test article treated cultures (both treatment conditions) exceeded the normal ranges.

Statistically significant linear trends (p≤0.001) were observed following both

treatment conditions in the absence of S-9.

Treatment of cells for 3+21 hours in the presence of S-9 resulted in frequencies of

MNBN cells that were significantly higher (p≤0.05 or p≤0.01), compared to those

observed in concurrent vehicle control cultures, for the lowest and highest

concentrations analysed (500 and 2000 μg/mL), with a statistically significant linear

trend (p≤0.05). However, these increases were small in magnitude such that the

MNBN cell frequency of only a single culture at the highest concentration analysed

(2000 μg/mL, inducing 31% cytotoxicity) marginally exceeded the normal range

(1.30% in comparison to the normal range of 0 to 1.20%). Overall these small

statistical increases were considered to present weak evidence of a test article related

effect under this test condition.

Mechanistic (FISH) Analysis

Investigative analysis, via the use of fluorescence in situ hybridisation (FISH) with

pan-centromeric DNA probes, was conducted to determine the underlying mechanism

of micronuclei formation in MNBN cells, i.e. whether chromosome loss (aneuploidy)

or chromosome breakage (clastogenicity) was the predominant mechanism of action.

Freshly prepared slides from stored cell pellets were processed as follows for

mechanistic analysis (test article concentrations / cultures selected had demonstrated

an indication of increased frequency of MNBN cells).

For the positive control compounds, treatment of cells with MMC (0.30 μg/mL) for

3+21 hours in the absence of S-9 induced 90% centromere-negative micronuclei

(C- MN) and 10% centromere-positive micronuclei (C+ MN) from a total of 100 MN

analysed, indicating a predominantly clastogenic mechanism. Treatment with VIN

(0.04 μg/mL) for 24+24 hours in the absence of S-9 induced 48% C- MN and

52% C+ MN from a total of 101 MN analysed, indicating a slightly predominant

aneugenic mechanism, although the ratio was close to 50:50.

Treatment with Tris(nitrato-O)nitrosylruthenium for 3+21 hours in the absence of S-9

at 2000 μg/mL induced 84% C- MN and 16% C+ MN from a total of 98 MN

analysed.

Treatment with Tris(nitrato-O)nitrosylruthenium for 24+24 hours in the absence of

S-9 at 200, 400 and 700 μg/mL induced 96%, 96% and 98% C- MN (4%, 4% and 2%

C+ MN), respectively, from a total of 100 MN analysed at each concentration.

The FISH analysis data for the 3+21 hour and 24+24 hour treatments in the absence

of S-9 confirm a predominantly clastogenic (chromosome breakage) mechanism for

both treatment conditions.

It is concluded that Tris(nitrato-O)nitrosylruthenium induced increases in the

frequency of micronuclei following treatments in cultured human peripheral blood

lymphocytes in the absence and presence of an Aroclor-induced rat liver metabolic

activation system (S-9). The maximum concentrations analysed for micronuclei were

either 2000 μg/mL (the maximum concentration recommended by the regulatory test

guidelines for the in vitro micronucleus assay) for the 3+21 hour treatments in the

absence or presence of S-9 or were limited by cytotoxicity for the 24+24 hour

treatments in the absence of S-9.

The use of FISH with pan-centromeric DNA probes demonstrated that for the

3+21 hour and 24+24 hour -S-9 treatments, micronuclei were generated via a

predominantly clastogenic mechanism (chromosome breakage) following treatment

with Tris(nitrato-O)nitrosylruthenium.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

The use of FISH with pan-centromeric DNA probes in the in vitro HLM assay (according to OECD487) demonstrated that for the 3+21 hour and 24+24 hour -S-9 treatments, micronuclei were generated via a predominantly clastogenic mechanism (chromosome breakage) following treatment with Tris(nitrato-O)nitrosylruthenium.

The data of Ballantyne (2020) show an uncommon response pattern with a positive response in strain TA102 only. In a follow-up test, Ballantyne (2021) tests both strain TA102 and E. coli strain WP2 uvrA under comparable experimental settings. Although the former strain confirms the positive response, the latter is clearly negative. This strongly suggests that the likely mechanisms for the positive response in strain TA102 is linked to ROS generation (strains TA98, TA100, TA1535, TA1537 are usually resistant to ROS). This mechanism is unlikely to lead to a positive response in in vivo mammalian testing due to the high ROS scavenging potential. In the testing proposal, an alternative strategy to avoid in vivo follow-up testing is proposed, next to direct in vivo follow-up testing. 

Justification for classification or non-classification

Based on the positive findings in the AMES (only strain TA102!) and in vitro HLM assays, and after having gained advise from an independent expert in genetic toxicity, the registrants have performed an additional in vitro AMES test with E. coli WP2 uvr1 as test strain (next to Salmonella strain TA102) to clarify the observation of the first AMES test (Ballantyne, 2020) and the potential role of ROS in the observed in vitro responses. Test data confirm the likely role of ROS as explaining factor for the observed responses in the in vitro assays. A proposal for follow-up testing (either via the generation of in vitro test data using modified protocols or directly via in vivo testing) is included in the dossier. Because of the likely dominant role of ROS as explanatory factor in the observed in vitro positive responses, the registrants consider a classification for mutagenicity currently not warranted. This position will be reconsidered as soon as proper follow-up testing has been performed.