Indium

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:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

see section 13 in IUCLID for read-across justification report

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2012

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (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:

Indium trihydroxide

EC Number:

243-947-7

EC Name:

Indium trihydroxide

Cas Number:

20661-21-6

Molecular formula:

H3InO3

IUPAC Name:

indium trihydroxide

Test material form:

solid: particulate/powder

Details on test material:

Name: Indium trihydroxide
Synonym: Indium(III) hydroxide
Batch/Lot number: OH-1306
Molecular formula: In(OH)3
Molecular weight: 165.84
Appearance: White powder
Purity: 72.9% indium
Expiry date: 02 November 2013
Storage conditions: Room temperature (15-30oC)
Safety Precautions: Routine safety precautions (lab coat, gloves, safety glasses and face mask) for unknown materials were applied to assure personnel health and safety.

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Elevage Janvier - Route des Chènes Secs B.P. 4105, 53940 LE GENEST-ST-ISLE, France
- Age at study initiation: 7 weeks
- Weight at study initiation:
36.5 - 38.8 g (males, preliminary experiment)
28.0 - 29.0 g (females, preliminary experiment)
32.8 – 36.7 g (males, main test)
- Fasting period before study: not reported
- Housing: Group caging (5 animals/cage or 2 animals/cage) ; Cage type: II. type polypropylene/polycarbonate
- Diet: ssniff® SM R/M-Z+H "Autoclavable complete feed ad libitum
- Water: tap water from municipal supply ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.4 – 24.9°C
- Humidity (%): 31 – 70 %
- Air changes (per hr): 15 – 20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 h/12 h

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: phosphate buffered saline (PBS: pH 7.2, Sigma, St. Louis, MO, USA)
- Justification for choice of solvent/vehicle: dissolvable in PBS

- Vehicle(s)/solvent(s) used: 1 % Methyl cellulose
- Justification for choice of solvent/vehicle:Based on the result of a preliminary solubility test, the test item was dissolved in 1 % Methyl cellulose for the treatment.
- Concentration of test material in vehicle: The concentration of the test item formulation was chosen to assure the same dosing volumes in mice for all dose levels (10 mL/kg bw). The test item was used for treatment in the main test at concentrations of 200, 100 and 50 mg/mL.
- Amount of vehicle (if gavage or dermal): 1 %
- Lot/batch no. (if required): O16147824

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was used for treatment in the main test at concentrations of 200, 100 and 50 mg/mL. The formulations were prepared immediately before the treatment in the Central Dispensary Unit of CiToxLAB Hungary Ltd.

Duration of treatment / exposure:

Animals are treated with the test substance once. Samples of bone marrow are taken, starting not earlier than 24 hours after treatment, but not extending beyond 48 hours after treatment with appropriate interval(s) between samples

Frequency of treatment:

once

Post exposure period:

24 h -48h

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 animals per dose; two additional male mice were dosed in high dose group (2000 mg/kg body weight) to replace any animals which die before the scheduled sacrifice time. Bone marrow smears were not prepared from additional mice as they were not used as replacement.

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide was used as the positive control administered by oral gavage dissolved in sterile physiological saline solution at a dose of 60mg/kg

Examinations

Tissues and cell types examined:

Polychromatic erythrocytes (PCE) and normochromatic erythrocytes (= normocytes, NCE) from the bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Based on the results of the preliminary toxicity test, 2000 mg/kg body weight (the maximum recommended dose) was selected for the highest dose level of the test item in the main micronucleus test. Two lower dose levels separated by a factor of two (1000 and 500 mg/kg body weight) were also included in the main test

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
- treatmetn: once gavage
- sacrifice: 24 h (all groups: low , mid and high dose) and 48h (only high dose) after the treatment

DETAILS OF SLIDE PREPARATION:
The bone marrow was flushed out of each pair of femurs with foetal bovine serum (5 mL) using a syringe and needle into a sterile centrifuge tube. After mixing, the cell suspension was concentrated by a gentle centrifugation and the supernatant was discarded. Smears of the cell pellet were made on standard microscope slides (2 slides / animal). Slides were air-dried at room temperature for approximately 24 hours.

Dried slides were fixed in methanol for a minimum of 5 minutes and allowed to air-dry. Two slides per animal were stained with 10 % Giemsa solution for 20 minutes then thoroughly rinsed with distilled water, and then air-dried at room temperature for at least 12 hours. After staining, coverslips were mounted on them.


METHOD OF ANALYSIS:
- Evaluation of two thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the incidence of the micronucleated cells. The frequency of micronucleated cells was expressed as percent of micronucleated cells based on the first 2000 PCEs counted in the optic field. Fewer than 2000 PCEs may be counted in the event of a clear positive effect.
-The proportion of immature among total erythrocytes was determined for each animal by also counting mature erythrocytes (NCEs) until a total of 1000 PCEs+NCEs had been observed

Evaluation criteria:

Criteria for Identification of Micronucleated Erythrocytes:

-A bluish mauve strongly coloured uniform round or oval particle in the cell.
-The particle should be large enough for the colour to be recognisable, and it should be located inside the cells. Areas with micronucleus-like particles outside the cells should not be used for analysis.
-During focusing, the particle should stay uniform in colour /light refraction and shape within a large interval and focus in the same plane as the erythrocyte.
-The unit of damage is deemed to be the cell, and therefore cells with two or more micronuclei will be counted as single micronucleated cells.

Statistics:

Kruskal Wallis test:
No biologically or statistically significant increases in the frequency of micronucleated polychromatic erythrocytes were seen in mice treated with the test item, compared to the negative control values at any of the sampling time points. The positive control treatment caused a large, clearly positive response demonstrating the sensitivity of the test system.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

No mortality or signs of systemic toxicity

Vehicle controls validity:

valid

Negative controls validity:

not examined

Remarks:

valid historical laboratory control database

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000, 1000, 500 and 250 mg/kg body weight
- Clinical signs of toxicity in test animals: All animals were free of clinical signs in the preliminary experiment except of one male in the 500 mg/kg body weight dose group showing piloerection. Therefore, the main test will be performed using male animals only because the toxic effect of the test item was similar in both sexes in the preliminary toxicity test.
- Evidence of cytotoxicity in tissue analyzed: not determined since no bone marrow smears prepared

RESULTS OF DEFINITIVE STUDY
-No marked effect of test item treatment on the body weight of the mice was observed in the main test

-No mortality or signs of systemic toxicity were observed during the study. In the low dose group (500 mg/kg body weight), piloerection was observed for one animal 24 hours after the treatment. The animals in the high and mid dose groups, furthermore in the negative (vehicle) control and positive control groups were symptom-free during the whole observation period

-Two thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells. The frequency of micronucleated cells was expressed as percent of micronucleated cells based on the first 2000 PCEs counted in the optic field. The proportion of immature among total erythrocytes was determined for each animal by also counting mature erythrocytes (NCEs) until a total of 1000 PCEs+NCEs had been observed.

The group treated with 1000 mg/kg bw, which gave the highest number of micronuclei at 24h, were compared with the vehicle control group using the Kruskal Wallis test. This gave a value of H = 0.185. Comparison of the vehicle control data and the high dose of the test item (2000 mg/kg body weight) at 48h gave a value of H = 0.124. Both H values are non-significant, giving a negative response.

The positive and negative control results were also compared, and gave a value of H = 6.863 (p<0.01). The positive control treatment therefore caused a very substantial increase, demonstrating the sensitivity of the test system.

The positive and negative control data are considered to give adequate data to confirm the validity of the study.

The frequency of micronucleated polychromatic erythrocytes of the negative (solvent) control group was within the range of historical laboratory control data; the positive control item produced a biologically and statistically relevant increase in the number of micronucleated polychromatic erythrocytes; each treated and control group included at least 5 analysable animals; therefore, the test was considered to be valid.

Any other information on results incl. tables

- No marked effect of test item treatment on the body weight of the mice was observed in the main test

-No mortality or signs of systemic toxicity were observed during the study. In the low dose group (500 mg/kg body weight), piloerection was observed for one animal 24 hours after the treatment. The animals in the high and mid dose groups, furthermore in the negative (vehicle) control and positive control groups were symptom-free during the whole observation period

-Two thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells. The frequency of micronucleated cells was expressed as percent of micronucleated cells based on the first 2000 PCEs counted in the optic field. The proportion of immature among total erythrocytes was determined for each animal by also counting mature erythrocytes (NCEs) until a total of 1000 PCEs+NCEs had been observed.

The group treated with 1000 mg/kg bw, which gave the highest number of micronuclei at 24h, were compared with the vehicle control group using the Kruskal Wallis test. This gave a value of H = 0.185. Comparison of the vehicle control data and the high dose of the test item (2000 mg/kg body weight) at 48h gave a value of H = 0.124. Both H values are non-significant, giving a negative response.

The positive and negative control results were also compared, and gave a value of H = 6.863 (p<0.01). The positive control treatment therefore caused a very substantial increase, demonstrating the sensitivity of the test system.

The positive and negative control data are considered to give adequate data to confirm the validity of the study.

The frequency of micronucleated polychromatic erythrocytes of the negative (solvent) control group was within the range of historical laboratory control data; the positive control item produced a biologically and statistically relevant increase in the number of micronucleated polychromatic erythrocytes; each treated and control group included at least 5 analysable animals; therefore, the test was considered to be valid.

Applicant's summary and conclusion

Conclusions:

No induction of micronuclei in bone marrow erythrocytes was observed following administration of Indium trihydroxide to mice at up to and including 2000 mg/kg bw/day; thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study.

Executive summary:

A study was conducted to determine whether Indium trihydroxide test item caused genotoxic effects resulting in the formation of micronuclei in erythrocytes of treated mice in accordance with the OECD guideline “Mammalian Erythrocyte Micronucleus Test”, No. 474 (1997).

No induction of micronuclei in bone marrow erythrocytes was observed following administration of Indium trihydroxide to mice at up to and including 2000 mg/kg bw/day; thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study.