Tris(nitrato-O)nitrosylruthenium

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study planned

Study period:

following ECHA approval

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out
tris(nitrato-O)nitrosylruthenium

- Name of the substance for which the testing proposal will be used [if different from tested substance]
tris(nitrato-O)nitrosylruthenium

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION :
- Available GLP studies
Ballantyne (2020). Ames test; According to OECD guideline 471; GLP compliant; positive with and without metabolic activation in histidine-requiring strain (TA102) of Salmonella typhimurium.
Ballantyne (2021). Ames test (one histidine-requiring strain (TA102) of Salmonella typhimurium and one tryptophan-requiring strain (WP2 uvrA) of Escherichia coli only); According to OECD guideline 471; GLP compliant; positive with and without metabolic activation in histidine-requiring strain (TA102) of Salmonella typhimurium.
Chirom (2020). In vitro micronucleus test; According to OECD guideline 487; GLP compliant; positive with and without metabolic activation.

- Available non-GLP studies
none

- Historical human/control data
No substance-specific data identified.

- (Q)SAR
It is acknowledged that the results of QSAR modelling are of very limited applicability for inorganic substances (e.g. metals) and organometallics.

- In vitro methods
The available in vitro methods have been considered as part of the tiered approach to testing (see “List of substance-specific available GLP and non-GLP studies considered for this test proposal” above for details). Further in vitro testing (e.g. for gene mutation in mammalian cells) is considered unnecessary given the available in vitro positive results.
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). A possible route to demonstrate absence of genotoxic action of tris(nitrato-O)nitrosylruthenium (other than indirectly via ROS generation) is a repeat of the in vitro micronucleus assay (Chirom 2020) and an additional in vitro gene mutation assay in mammalian cells (hprt-assay; OECD476), both in presence of antioxidants. If both assays would show a negative response, strong in vitro evidence is available showing that the likely mechanism of genotoxic action in the in vitro assays is via ROS generation. Since this mechanism is unlikely to lead to a positive response in in vivo mammalian testing due to the high ROS scavenging potential, this in vitro testing can be used in a weight-of-evidence argumentation to avoid in vivo testing. However, the registrants are unsure on the acceptability of this in vitro testing strategy by the ECHA (given the positive responses in the standard AMES and in vitro MN assays), and the possibility for a request of a confirmatory in vivo testing irrespective of the outcome of any potential further in vitro testing. Also, we are unsure if we can successfully reduce all of the ROS effects with the modified in vitro assays, and the selection and validation of antioxidants might be an expensive and time consuming project without any certainty on possible acceptance. As such, direct in vivo testing is proposed to obtain a conclusive answer to the positive in vitro findings.

- Weight of evidence
Tris(nitrato-O)nitrosylruthenium induced mutation in histidine requiring strain TA102 of Salmonella typhimurium in the absence and presence of metabolic activation. When tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two female donors, 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 use of FISH with pan-centromeric DNA probes demonstrated that micronuclei were generated via a predominantly clastogenic mechanism (chromosome breakage). No in vivo genotoxicity studies were identified for Tris(nitrato-O)nitrosylruthenium.

- Grouping and read-across
No critical supplementary read-across data were identified.

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
The substance is not classified for carcinogenicity or mutagenicity therefore genetic toxicity testing cannot be waived. As outlined in the Integrated Testing Strategy (ITS) for mutagenicity (ECHA, 2015), “if there is a positive result in any of the in vitro studies from Annex VII or VIII and there are no appropriate results available from an in vivo study already, an appropriate in vivo somatic cell genotoxicity study should be proposed.” No in vivo genotoxicity data were identified for this substance. Moreover, there are no adaptations for in vivo somatic cell genotoxicity testing according to Column 2 of the REACH Annexes on information requirements (EC, 2014). Hence, the observation of mutagenic activity in both bacteria and mammalian cells necessitates the consideration of further in vivo testing “as a last resort” (ECHA, 2016).

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed
In order to assess the potential to induce genotoxicity in vivo, an alkaline comet assay (OECD Test Guideline 489), with a concomitant micronucleus assay and combined toxicokinetic assessment is proposed. The purpose of the comet assay is to identify substances that cause DNA damage, by detecting single and double stranded breaks. “These strand breaks may be repaired, resulting in no persistent effect, may be lethal to the cell, or may be fixed into a mutation resulting in a permanent viable change. They may also lead to chromosomal damage which is also associated with many human diseases including cancer” (OECD, 2016). It is proposed that comet measurements should be taken in both the glandular stomach and duodenum (site-of-contact tissues) and liver (due to the apparent influence of S9 metabolic activation in vitro), in rats following oral dosing. Bone marrow is selected as the target tissue for micronuclei assessment. Inclusion of a parallel toxicokinetic study is proposed for the purpose of demonstrating that adequate target tissue exposure to the test substance has been achieved. It is proposed to test Tris(nitrato-O)nitrosylruthenium

References:
ECHA (2015). European Chemicals Agency. Guidance on Information Requirements and Chemical Safety Assessment. Chapter R.7a: Endpoint specific guidance. Version 4.1. October 2015. https://echa.europa.eu/documents/10162/13632/information_requirements_r7a_en.pdf
ECHA (2016). European Chemicals Agency. How to prepare registration and PPORD dossiers. Version 1.0. April 2016. https://echa.europa.eu/documents/10162/22308542/manual_regis_and_ppord_en.pdf
EC (2014). European Commission. C1 Regulation (EC) No 1907/2006 of the European parliament and of the council of 18 December 2006 concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), establishing a European Chemicals Agency, amending Directive 1999/45/EC and repealing Council Regulation (EEC) No 793/93 and Commission Regulation (EC) No 1488/94 as well as Council Directive 76/769/EEC and Commission Directives 91. http://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:02006R1907-20140822
OECD (2016). Organisation for Economic Cooperation and Development. OECD guideline for the testing of chemicals. In Vivo Mammalian Alkaline Comet Assay. TG 489. Adopted: 29 July 2016. http://www.oecd-ilibrary.org/docserver/download/9716431e.pdf?expires=1475142425&id=id&accname=guest&checksum=151E9EE218B896F623F1DAE23EB3667F

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

GLP compliance:

yes

Type of assay:

other: single cell gel/comet assay in rodents for detection of DNA damage

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

Test animals

Species:

rat

Details on test animals or test system and environmental conditions:

Inclusion of a parallel toxicokinetic study is proposed for the purpose of demonstrating that adequate target tissue exposure to the test substance has been achieved.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil