Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: other: chromosome damage
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 August - 6 September 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Conducted according to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Reference substance name:
Rhodium(III) nitrate hydrate
EC Number:
603-842-2
Cas Number:
13465-43-5
Molecular formula:
H4N3O11Rh
IUPAC Name:
Rhodium(III) nitrate hydrate
Details on test material:
- Name of test material (as cited in study report): rhodium (III)-nitrate-hydrate
- Substance type: brown powder
- Physical state: solid
- Purity: no data
- Composition of test material, percentage of components: 34.57% rhodium
- Lot/batch No.: 10106
- Expiration date of the lot/batch: stable until 08.09.2007
- Storage condition of test material: at room temperature
- Other: hygroscopic

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann
- Age at study initiation: 6-12 weeks (minimum of 7 weeks old at start of treatment)
- Weight at study initiation: males 28.2-31.7 g, females 24.5-29.4 g
- Assigned to test groups randomly: yes
- Housing: 5 animals of identical sex/Macrolon Type III cage with Lignocel bedding
- Diet: Altromin 1324 maintenance diet for rats and mice, totally-pathogen-free, frequency of administration not specified
- Water: ad libitum
- Acclimation period: “adequate acclimatisation period”

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 55 ± 10
- Photoperiod (hrs dark / hrs light): artificial light 06:00 – 18:00

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: 0.9% sodium chloride
- Justification for choice of solvent/vehicle: relatively non-toxic for the animals
Details on exposure:
No data
Duration of treatment / exposure:
Single injection.
Frequency of treatment:
once
Post exposure period:
44 hrs (for all groups) and 68 hrs (for negative control and highest dose group only)
Doses / concentrations
Remarks:
Doses / Concentrations:
16, 40 and 80 mg/kg bw given in a single volume of 10 mL/kg bw.
Basis:
other: Covering a range of doses from the maximum tolerable dose (ascertained in a pre-experiment) to slight toxicity.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
yes, historical
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): no data
- Route of administration: intraperitoneal
- Doses / concentrations: 40 mg/kg bw given as a single dose in a volume of 10 mL/kg bw

Examinations

Tissues and cell types examined:
Peripheral blood
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: 0.2, 0.5 and 1 x the maximum tolerable dose obtained from a pre-experiment for toxicity

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): animals treated once and the blood sampled from the tail vein 44 hrs (all groups) and 68 hrs (highest dose group and negative control) after treatment

DETAILS OF SLIDE PREPARATION: Blood cells were immediately fixed in ultracold methanol, washed, at least 16 hrs after fixation, in Hank’s balanced salt solution and centrifuged at 600 g for 5 min. The supernatant was discarded and the blood cell populations were discriminated using specific antibodies against CD71 and CD61 and the DNA content of the micronuclei was determined by the use of propidium iodide, a DNA specific stain

METHOD OF ANALYSIS: Evaluation was performed using a flow cytometer. Anti-CD71 antibodies were labelled with fluoresceinisothiocyanate and anti-CD61 antibodies were labelled with phycoerythrin. Particles were differentiated using Forward Scatter and Side Scatter parameters of the flow cytometer and fluorescence intensity was recorded. At least 10000 immature erythrocytes/animal were scored for the incidence of micronucleated immature erythrocytes. The ratio between immature and mature erythrocytes was determined to detect an eventually occurring cytotoxic effect of the test material and the results were expressed as relative PCE (proportion of polychromatic (immature) erythrocytes among total erythrocytes).
Evaluation criteria:
A positive result is determined by a:
- dose-related increase in the number of micronucleated cells and/or
- biologically relevant increase in the number of micronucleated cells for at least one of the dose groups.

The biological relevance as well as the statistical significance of the results are the criterion for interpretation and a test item is considered to be negative if there is no biologically relevant and/or statistically significant increase in the number of micronucleated cells at any dose level.

The data generated are considered acceptable if:
- the weight variation of the animals at the start of the study is minimal and does not exceed ±20% of the mean weight of each sex,
- the background frequency of micronucleated cells is in the normal range as reported in the literature or is within the laboratory’s historical range and
- the test system is sensitive to the known mutagen as judged by the results in the concurrent positive control animals.
Statistics:
The non-parametric Mann-Whitney test was used.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 25 (1 male), 80 (3 males and 3 females), 125 (1 male), 500 (1 male and 1 female), 1000 (1 female), 2000 (1 female) mg/kg bw
- Solubility: administered as 10 mL/kg bw
- Clinical signs of toxicity in test animals: Reduction of spontaneous activity and rough fur was seen at 25 mg/kg bw, with slight cramps and palpebral closure also being seen at 80 mg/kg bw. The mice receiving 125, 500 and 1000 mg/kg bw were euthanised 24, 30 and 48 hrs after treatment respectively in consideration of animal welfare reasons. At the top dose level, the mouse died within 23 hrs of the intraperitoneal injection.
- Evidence of cytotoxicity in tissue analyzed: no data
- Rationale for exposure: “According to the OECD guideline 474”. The selection of the highest dose was conducted in accordance with OECD guidelines 420 and 423, particularly with respect to selection of dose spacing and animal welfare aspects. The volume to be administered was compatible with the physiological space available.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): The mean values of micronuclei observed for the negative control at 44 hrs were 0.25 and 0.22% for males and females respectively and at 68 hrs were 0.21 and 0.19% respectively. The mean values for the positive control after 44 hrs were 2.31 and 1.46% in the males and females respectively. In the treated mice, the mean values after 44 hrs were 0.34, 0.4 and 0.31% in the males and 0.31, 0.26 and 0.29% in the females treated with 16, 40 and 80 mg/kg bw respectively. After 68 hrs, the mean values observed in the highest treated group were 0.21 and 0.19% for the males and females respectively.
- Appropriateness of dose levels and route: Three dose levels covering a range from the maximum tolerable dose, as determined in a range-finding experiment, down to slightly toxic were used.
- Statistical evaluation: Slight increases in the percentage of cells with micronuclei compared to the corresponding vehicle controls were noted at the lowest test dose level in both sexes, in the mid-dosed female group and in both sexes at the top-dose level after 44 hrs but these were not found to be statistically significant. In the mid-dosed male group, the mean value was significantly increased when compared with the vehicle control but this was within the range of the historical control data. After 68 hrs, the values were comparable with the controls. Using the non-parametric Mann-Whitney Test, no statistically significant enhancement (p<0.05) of cells with micronuclei was noted in the treated groups, except for the male mid-dosed group. However, these values were within the range of the historical control data of the vehicle control.

Any other information on results incl. tables

Toxic effects were noted in all of the animals treated with 40 and 80 mg/kg bw and included a reduction in spontaneous activity, cramps, palpebral closure and rough fur. Only one male in the low dosed group showed similar toxic effects.

 

The mean values noted for the relative PCE in the vehicle control animals after 44 hrs were 1.73 for the males and 1.43 for the females and after 68 hrs were 2.16 and 1.97 for the males and females respectively, which were within the range of the historical control data. The mean values of the relative PCE in the low-, medium- and high-dosed groups were 1.56, 1.63 and 1.44 for males and 1.66, 1.74 and 1.32 for females respectively after 44 hrs and 1.82 and 2.12 for the high-dosed males and females after 68 hrs respectively. No statistically significant effects were noted and the mean values observed were all within the range of the historical control data of the vehicle control.

Applicant's summary and conclusion

Conclusions:
Rhodium trinitrate hydrate failed to induce a biologically relevant, or dose-related, increase in the incidence of micronuclei in the immature erythrocytes of the bone marrow of mice following administration by intraperitoneal injection at up to 80 mg/kg bw.
Executive summary:

Rhodium trinitrate hydrate was studied for the ability to induce micronuclei in bone marrow cells (polychromatic erythrocytes; PCE) in mice, in a study conducted in accordance with OECD Test Guideline 474, and to GLP. Mice (5/sex/group) received rhodium trinitrate hydrate by intraperitoneal injection (in a volume of 10 mL/kg bw) at a single dose of 16, 40 and 80 mg/kg bw (the maximum tolerable dose) and peripheral blood samples were collected and analysed for the induction of micronuclei 44 and 68 hrs after treatment. The non-parametric Mann-Whitney Test was used, but both biological relevance and statistical significance were considered. At least ten thousand cells per animal were scored for micronuclei and the ratio of polychromatic to mature cells (relative PCE) was calculated as a measure of toxicity.

Only one male in the low-dose group showed signs of slight systemic toxicity (up to 2 hours after treatment) whereas all animals in the mid- and high-dosed groups showed adverse effects (for up to 24 hours). The relative PCE of any of the test dose groups was not statistically significantly different from the vehicle controls, whereas the incidence of micronucleated immature erythrocytes was statistically significantly increased in the mid-dosed males after 44 hrs. However, as this increase was within the range of the historical control data of the vehicle control and the other mean values showed no statistical significance over the control values, this effect was regarded as not biologically relevant.

It is concluded that rhodium trinitrate hydrate did not increase the incidence of micronuclei in mice after a single intraperitoneal dose of up to 80 mg/kg bw (Becker et al., 2007).