Hexakis[μ-(acetato-O:O')]-μ3-oxo-triangulo-triruthenium acetate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16-Jan-1997 to 10-Feb-1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: OECD Guideline study, to GLP, with acceptable deviation (only 1000 PCEs/animal scored for micronuclei)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

(only 1000 PCEs/animal scored for micronuclei)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Hexakis[μ-(acetato-O:O')]-μ3-oxo-triangulo-triruthenium acetate

EC Number:

259-653-7

EC Name:

Hexakis[μ-(acetato-O:O')]-μ3-oxo-triangulo-triruthenium acetate

Cas Number:

55466-76-7

Molecular formula:

C12H18O13Ru3.C2H3O2

IUPAC Name:

hexamethyl-2λ³-oxa-4λ³-oxa-6λ³-oxa-8λ³-oxa-10λ³-oxa-12λ³-oxa-13λ³-oxa-15λ³-oxa-16λ³-oxa-18λ³-oxa-19λ³-oxa-21λ³,22λ¹-dioxa-1,5,9-triruthenahexacyclo[7.3.3.3¹,⁵.3⁵,⁹.1¹,⁵.0⁹,²²]docosa-3,7,11,14,17,20-hexaene-1,1,1,5,5,5,9,9,9-nonakis(ylium)-2,6,10,13,16,19-hexaide-22,22-diuide acetate

Details on test material:

- Name of test material (as cited in study report): ruthenium acetate
- Substance type: no data
- Physical state: dark green/black crystalline solid
- Analytical purity: no data
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: 60390
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: room temperature
- Other:
- Receipt date: 16-Aug-1996

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate, Kent
- Age at study initiation: 5-7 weeks
- Weight at study initiation: males 24-30 g, females 21-26 g
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: <=7/cage, solid floor polypropylene cages with woodflakes bedding
- Diet (e.g. ad libitum): Rat and Mouse Expanded Diet No. 1, ad libitum
- Water (e.g. ad libitum): mains water, ad libitum
- Acclimation period: >=5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-21
- Humidity (%): 47-52
- Air changes (per hr): ~15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 16-Jan-1997 To: 10-Feb-1997

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: physiol. saline
- Justification for choice of solvent/vehicle: no data, but well-known
- Concentration of test material in vehicle: 5, 10 and 20 mg/ml (for 50, 100 and 200 mg/kg bw doses respectively)
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw
- Type and concentration of dispersant aid (if powder): not applicable
- Lot/batch no. (if required): Gibco 18Q7563
- Purity: no data

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- freshly prepared as required as a suspension in the vehicle

Duration of treatment / exposure:

single intraperitoneal administration

Frequency of treatment:

single administration

Post exposure period:

24, 48 and 72 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

5 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

10 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

20 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5/sex per dose at 24-hour timepoint;
also 5/sex at top dose at 48- and 72-hour timpoints

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): "known to produce micronuclei under the conditions of the test"
- Route of administration: oral
- Doses / concentrations: 50 mg/kg bw

Examinations

Tissues and cell types examined:

erythrocytes from bone marrow of femur

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: based on range-finding study in which deaths were observed with doses of 250-2000 mg/kg bw, but not at 200 mg/kg bw or below

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): sampled at 24, 48 and 72 hours after dosing in top dose group (200 mg/kg bw) and at 24 hours in mid and low dose groups (100 and 50 mg/kg bw)

DETAILS OF SLIDE PREPARATION:
- immediately after sacrifice, 1 femur dissected/animal, aspirated with foetal calf serum
- centrifuged, resuspended and bone marrow smears prepared
- air-dried, fixed in absolute methanol, stained with May-Grunwald/Giemsa

METHOD OF ANALYSIS:
- slides coded prior to examination
- light microscopy at 1000x magnification
- incidence of micronucleated cells/1000 polychromatic erythrocytes (PCEs; blue-stained, immature cells) scored for each animal
- incidence of normochromatic erythrocytes (NCEs; pink-stained, mature cells)/1000 erythrocytes scored for each animal
- incidence of micronuclei in these NCEs scored for each animal
- ratio of PCE:NCE calculated

Evaluation criteria:

Positive mutagenic response: statistically significant increase in number of micronucleated PCEs at any timepoint
Positive bone marrow toxicity: statistically significant decrease in group mean PCE:NCE ratio

Statistics:

Data transformed, then analysed by 2-tailed Student's t-test; significant results confirmed using one-way analysis of variance (United Kingdom Environmental Mutagen Society (UKEMS) Sub-committee on Guidelines for Mutagenicity Testing Report, part III, 1989)

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Remarks:

at all doses at all timepoints

Toxicity:

yes

Remarks:

clinical signs of toxicity at top dose at all timepoints; bone marrow toxicity at top dose at 48-hour timepoint

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range:
- intraperitoneal: 125-2000 mg/kg bw
- oral: 1000-2000 mg/kg bw
- Solubility: administered as a suspension
- Clinical signs of toxicity in test animals:
- intraperitoneal: deaths at 250 mg/kg bw (1/2), 500 mg/kg bw (2/2), 1000 mg/kg bw (2/2) and 2000 mg/kg bw (2/2); clinical signs (including hunched posture, lethargy, decreased respiratory rate, laboured respiration, ataxia, ptosis and grey extremities) at 1000 and 2000 mg/kg bw
- oral: no deaths, no clinical signs
- Evidence of cytotoxicity in tissue analyzed: not examined in range-finding study
- Rationale for exposure: for the main study, the doses selected were the maximum tolerated dose of 200 mg/kg bw and 2 lower doses
- Harvest times: not examined in range-finding study
- High dose with and without activation: not applicable to in vivo study

RESULTS OF DEFINITIVE (MAIN) STUDY
- Clinical signs of toxicity in test animals: in top dose group (200 mg/kg bw), clinical signs of toxicity at all timepoints, including hunched posture, ptosis, dehydration, decreased respiratory rate, laboured respiration, ataxia, emaciation, lethargy, piloerection and grey extremities; deaths in 72-hour group (males 1/5, females 3/5)
- Induction of micronuclei (for Micronucleus assay): see Table 1
- Ratio of PCE/NCE (for Micronucleus assay): see Table 1
- Appropriateness of dose levels and route: appropriate; highest dose produced signs of toxicity such that higher dose levels would be expected to produce lethality; highest dose also produced some indication of toxicity in the bone marrow (i.e. a reduction in the PCE:NCE ratio at 48 hours)
- Statistical evaluation: no statistically significant increase in the number of PCE with micronuclei per 1000 PCE at any dose at any timepoint in either sex; positive control produced a highly significant increase

Any other information on results incl. tables

Table 1:Results of in vivo micronucleus test with ruthenium acetate

(5 males and 5 females/group; mean ± standard deviation)

Treatment group		Number of PCE with micronuclei per 1000 PCE		PCE:NCE ratio
		Group mean	SD	Group mean	SD
Dose	Sampling time (hours)
Vehicle control	72	0.7	1.3	1.62	0.35
	48	0.8	0.9	1.53	0.67
	24	1.3	0.8	1.49	0.49
Positive control	24	28.9***	13.1	1.44	0.5
High dose (200 mg/kg bw)	72^a	0.3	0.5	1.25	0.93
	48	0.7	0.9	0.81*	0.52
	24	0.6	0.7	1.17	0.31
Mid dose (100 mg/kg bw)	24	0.5	0.5	1.55	0.39
Low dose (50 mg/kg bw)	24	0.6	0.7	1.33	0.53

 

PCE, polychromatic erythrocyte, NCE, normochromatic erythrocyte, SD, standard deviation, *** p<0.001, * p<0.05, a data from 6 animals

 

Applicant's summary and conclusion

Conclusions:

In a reliable in vivo study, conducted according to OECD Test Guideline 474 and to GLP, no genotoxicity was seen in the bone marrow of mice administered ruthenium acetate by intraperitoneal injection at up to 200 mg/kg bw; clinical signs of toxicity and toxicity in the bone marrow were seen at the highest dose.

Executive summary:

Groups of 5 male and 5 female mice were administered a single intraperitoneal dose of ruthenium acetate at 50, 100 or 200 mg/kg bw and evaluated for the incidence of micronuclei in the bone marrow 24 hours after dosing. Two further groups were administered 200 mg/kg bw and evaluated 48 and 72 hours after dosing. Vehicle control groups were evaluated at each timepoint and a positive control group was orally administered cyclophosphamide at 50 mg/kg bw and evaluated at 24 hours.

Clinical signs of toxicity were seen in all groups dosed with 200 mg/kg bw and 4/10 mice died in the 72 -hour group at this dose level. Bone marrow toxicity, indicated by a decrease in the ratio of normochromatic to polychromatic erythrocytes (NCE:PCE), was seen in the 48 -hour group. There was no increase in the incidence of micronuclei at any dose at any timepoint in either sex. A clear positive response was seen in the positive control group.

In a reliable GLP study, conducted according to OECD Test Guideline 474, no genotoxicity was seen in the bone marrow of mice administered ruthenium acetate by intraperitoneal injection at up to 200 mg/kg bw; clinical signs of toxicity and toxicity in the bone marrow were seen at the highest dose.