Rhodium chloride (RhCl3), hydrate

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:

12 February - 15 March 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Rhodium (III) chloride hydrate

IUPAC Name:

Rhodium (III) chloride hydrate

Constituent 2

Reference substance name:

20765-98-4, 13569-65-8

IUPAC Name:

20765-98-4, 13569-65-8

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Rhodium (III) chloride hydrate, solution
- Substance type: No data
- Physical state: Red liquid
- Analytical purity: No data
- Impurities (identity and concentrations): No data
- Composition of test material, percentage of components: 15.95% RhCl3 without water, 1.78% HCl
- Isomers composition: Not applicable
- Purity test date: No data
- Lot/batch No.: 103/05
- Expiration date of the lot/batch: 30 April 2007
- Stability under test conditions: No data
- Storage condition of test material: Room temperature

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winklemann
- Age at study initiation: 6-12 weeks at start of acclimatisation (minimum 7 weeks at start of treatment)
- Weight at study initiation: Males, 27-32; females, 23-28 g
- Assigned to test groups randomly: Yes
- Fasting period before study: No data
- Housing: 5 animals of the same sex per cage. Macrolon Type III (Hereto) cage with lignocel bedding.
- Diet (e.g. ad libitum): Altromin 1324 maintenance diet
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: One week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 55 ± 10
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: No data

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: Sodium chloride
- Justification for choice of solvent/vehicle: Solvent was chosen “according to its relative non-toxicity for the animals”.

Details on exposure:

The test item was prepared and diluted in 0.9% NaCl within 1 hour before treatment. All anmals received a single volume intraperitoneal injection of 10 mL/kg bw

Duration of treatment / exposure:

Acute exposure

Frequency of treatment:

Single dose

Post exposure period:

44 and (for the highest dose only) 68 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
200, 500 and 1000 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Justification for choice of positive control(s): No data
- Route of administration: Intraperitoneal
- Doses / concentrations: 40 mg/kg bw (10 mL/kg bw)

The solution is prepared on the day of administration.

Examinations

Tissues and cell types examined:

Erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Due to the results obtained in the range-finding study, 1000 mg/kg bw was chosen as the maximum tolerable dose (MTD). The MTD is defined as the dose producing signs of toxicity such as lethargy, palpebral closure, prone position etc. Higher doses are anticipated to produce lethality within 48 hr.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Single intraperitoneal treatment; exposition times were 44 hr for all dose groups and 68 hr for the negative control and the highest dose group.

DETAILS OF SLIDE PREPARATION: Blood cells obtained from the tail vein were immediately fixed in “ultracold” methanol, washed in Hank’s balanced salt solution, and centrifuged at 600 x g for 5 minutes (supernatant was subsequently discarded).

METHOD OF ANALYSIS: Evaluation of all samples performed using a flow cytometer at least 16 hr after fixation. At least 10,000 immature erythrocytes were evaluated for the presence of micronuclei from each animal. The total numbers of polychromatic (immature) erythrocytes among total erythrocytes (relative PCE [rel. PCE]) was calulated in order to detect a cytotoxic effect.

Evaluation criteria:

A positive result is considered as either a dose-related increase in the number of micronucleated cells and/or a biologically relevant increase in the number of micronucleated cells for at least one of the dose groups.

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.

Statistics:

Nonparametric Mann-Whitney Test (p<0.05)

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

positive

Toxicity:

yes

Remarks:

Observed at the highest two doses: effects at 1000 mg/kg bw included reduction of spontaneous activity, apathy, palpebral closure, rough fur and cramps. Slight toxicity was apparent at 500 mg/kg bw

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 400 (one female), 1000 (3 mice/sex) and 2000 (one female) mg/kg bw
- Solubility: No data
- Clinical signs of toxicity in test animals: No toxic symptoms were seen in one female mouse following a single intraperitoneal injection of 400 mg/kg bw. In mice (3/sex) receiving an injection of 1000 mg/kg bw, systemic toxicity was seen (including reduction of spontaneous activity, prone position, cramps, apathy, palpebral closure and/or rough fur), but all survived 72 hours after treatment. One female received 2000 mg/kg bw, and died six hours after exposure.
- Evidence of cytotoxicity in tissue analyzed: No data
- Rationale for exposure: No data
- Harvest times: 30 minutes, 5 hours, 1 day and 2 days
- High dose with and without activation: 2000 mg/kg bw

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): Not applicable
- Induction of micronuclei (for Micronucleus assay): All mean values of micronuclei formation following treatment with the test item were above the corresponding negative controls. A biologically relevant increase in the induction of micronucle was apparent. Further, this exhibited a dose-response relationship (see Table 1 for details).
- Ratio of PCE/NCE (for Micronucleus assay): The relative PCE was determined for each animal for the assessment of cytotoxicity. The negative control values noted at 44 hr were 2.13 for males and 1.57 for females while the analogous values at 68 hr were 2.67 and 2.62 respectively. These values were slightly above the range of the control data (1.38-2.08) as reported in the literature, aside from the 44-hr females. Relative PCE values for the treated groups were as follows (data for males and females respectively): 2.01 and 1.52 (0.2 MTD, 44 hr); 2.21 and 1.45 (0.5 MTD, 44 hr); 2.54 and 1.67 (1 MTD, 44 hr); 2.00 and 1.47 (1 MTD, 68 hr). These values were all within the range of the corresponding negative control.
- Appropriateness of dose levels and route: The dose levels were selected on the basis of the preliminary study
- Statistical evaluation: A statistically significant (p<0.05) increase in the frequency of blood cells with micronuclei was noted in the three test groups and the positive control group, when compared to the negative controls (see Table 2 for details).

Any other information on results incl. tables

Table 1: Percentage of blood cells with micronuclei

Dose group	Concentration (mg/kg bw)	Preparation time (h)	Male (%)	Female (%)
Vehicle control	0	44	0.31	0.20
Positive control	40	44	2.25	1.72
1 MTD	1000	44	2.57	2.48
0.5 MTD	500	44	1.65	1.52
0.2 MTD	200	44	0.90	0.97
Vehicle control	0	68	0.32	0.21
1 MTD	1000	68	0.73	0.56

MTD: Maximum tolerable dose

Table 2: Statistical significance at the 5% level (p<0.05)

Vehicle control versus test group	Preparation time (hr)	Significance		p-value
		Male	Female	Male	Female
Positive control	44	+	+	0.0079	0.0079
1 MTD	44	+	+	0.0159	0.0079
0.5 MTD	44	+	+	0.0079	0.0079
0.2 MTD	44	+	+	0.0317	0.0079
1 MTD	68	+	+	0.0159	0.0079

+: Significant

-: Not significant

The negative controls (24 and 48 hr) evaluated were within the range of the control data (0.15 -0.65%) reported in the literature.

The positive control (cyclophosphamide) induced a statistically significant increase in the induced micronucleus frequency (percentage of cells with micronuclei was 2.25 and 1.72% for males and females, respectively), thus demonstrating the validity of the assay.

The weight variation of treated animals (+/- 8.7 and 9.7% for males and females, respectively) did not exceed +/- 20% of the mean weight of each sex, thus fulfilling the data acceptance criteria.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): positive
In an in vivo guideline study, to GLP, rhodium (III) chloride hydrate solution induced a dose-related and statistically significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice (5/sex/dose) following a single intraperitoneal injection at 200, 500, and 1000 mg/kg bw, when compared to the vehicle controls.

Executive summary:

An in vivo mammalian erythrocyte micronucleus test (conducted according to OECD test guideline 474, and to GLP) was performed to assess the potential of rhodium (III) chloride hydrate, solution to induce micronuclei in the blood cells of mice.

 

Following a range-finding study, groups of young healthy mice (5/sex/dose) were administered the test material in sodium chloride solution at 0, 200, 500, and 1000 (the MTD) mg/kg bw by single intraperitoneal injection. Peripheral blood was obtained from the tail vein 44 hours and (at the highest dose only) 68 hours after treatment. At least 10,000 polychromatic (immature) erythrocytes (PCEs) were counted from each animal, and scored for the presence of micronuclei. Additionally, the proportion of PCEs among total erythrocytes was calculated for each animal (rel. PCE).

 

A biologically relevant and dose-related increase in the incidence of micronucleated polychromatic erythrocytes was observed in all treated groups compared to the concurrent vehicle controls. The rel. PCE values were in the range of the corresponding vehicle control. Clinical signs of toxicity were observed at the two highest doses. Cyclophosphamide (positive control), administered intraperitoneally at 40 mg/kg bw, induced a significant increase in micronucleus frequency, demonstrating the adequacy of the assay.

 

In conclusion, rhodium (III) chloride hydrate solution induced a dose-related and statistically significant increase in the frequency of micronuclei in erythrocytes of mice following a single intraperitoneal injection at 200, 500, and 1000 mg/kg bw, when compared to the vehicle controls.