Indium

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an in vitro bacterial mutation assay (Ames test, equivalent to OECD 471) indium did not induce any mutations in Salmonella typhimurium strains (TA1535, TA1537, TA98, TA100) and Escherichia Coli strain WP2uvrA pKM101 with and without metabolic activation (S9) (Asakura et al 2009).

In a chromosome aberrations test in Chinese hamster lung CHL/IU cells, equivalent to OECD 473, indium did not induce chromosome aberrations in vitro, both in the absence and presence of metabolic activation (Asakura, 2009)

In an OECD test Guideline 473 study, conducted to GLP, indium hydroxide did not induce chromosome aberrations in Chinese hamster lung fibroblasts (V79) in vitro, both in the absence and presence of metabolic activation (Hargitai, 2013)

In an OECD Test Guideline 476 mouse lymphoma assay, conducted to GLP, indium hydroxide did not induce a statistically and biologically significant increase in the mutant frequency at the TK +/_ locus in L5178Y mouse lymphoma cells either in the presence or absence of a rat metabolic activation system (S9 fraction) (Hargitai, 2013)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Remarks:

GLP compliance not specified in publication

Type of assay:

bacterial reverse mutation assay

Target gene:

not applicable

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

5,000; 1,250; 313; 78.1; 19.5; 4.88; 1.22 and 0.305 µg/plate (dose-finding test)
5,000; 2,500; 1,250; 625; and 313 µg/plate (main test)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Remarks:

solvent DMSO

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

With S9mix: 2-AA (TA 100; TA 1535, TA 1537, TA98 and WP2µvrA/pKM101. Without S9 mix: AF-2 (TA 100, TA 98 and WP2µvrA/pKM101), NaN3 (TA 1535), 9-AA (TA 1537)

Positive control substance:

9-aminoacridine

sodium azide

furylfuramide

other: 2-AA (2-aminoanthracene)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20min at 37°C


NUMBER OF REPLICATIONS: 3

Evaluation criteria:

The test substance was judged to be mutagnic (or positive) when the mean number of revertant colonies dose-dependently increased two-fold or more than that of the corresponding negative control for at least one tester strain with or without S9mix.

Statistics:

the data was not analyzed statistically

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

none

-dose-finding test: the numbers of revertant colonies were less than twice that of the corresponding negative (solvent) control in all tester strains with or without S9 mix.

-main test: the numbers of revertant colonies were less than twice that of the corresponding negative (solvent) control in all tester strains with or without S9 mix.

Conclusions:

The test material was considered to be non-mutagenic for all the used bacterial strains (Salmonella typhimurium as well as Escherichia coli) with as well as without metabolic activation

Executive summary:

A study was conducted to determine the potential mutagenicity of the test material using bacterial reverse mutation assay (e.g. Ames test). 

No significant increases in the frequency of revertant colonies were recorded at any dose level.

 

The test material was considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

not specified

Remarks:

GLP compliance is not specified in publication

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

mammalian cell line, other: CHL/IU cells

Details on mammalian cell type (if applicable):

purchased from Dainippon Pharmaceutical Co.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

-S9 mix: 156, 313, 625 and 1,250 µg/ml, + S9 mix: 625; 1,250; 2,500 and 5000 µg/ml (pulse treatment, main test)
- 125, 250, 500 and 1000µg/ml (continuous treatment 24h)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: CMC-Na

Untreated negative controls:

yes

Remarks:

sodium carboxymethylcellulose (CMC-Na solution)

Positive controls:

yes

Remarks:

mitomycin C (MMC) and Benzo(a)pyrene (BP)

Positive control substance:

benzo(a)pyrene

mitomycin C

Details on test system and experimental conditions:

Chromosome aberrations:

METHOD OF APPLICATION: in suspension

DURATION
- Preincubation period: 3 days
- Exposure duration: 6h, washed 3X and incubated for a further 18h (pulse treatment) ; 24h (continuous treatment)

SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): 3% Giemsa solution

NUMBER OF REPLICATIONS: two slides for each plate

NUMBER OF CELLS EVALUATED: 100 metaphase cells per plate (i.e 200 cells for each concentration)

Evaluation criteria:

a cell having at least one structural chromosomal aberration is classified as aberrant cell
Test substance was judged to have the potential to induce chromosomal abberation (positive: +)
if either,or both, of the aberration incidences of two types (structural or numerical) among oberserved cells was 10% or more.
if either, or both, of the two incidences were 5% or more and less than 10% the potential was judged inconclusive (+/-)
when both of the incidences were less than 5%, it was judged to be negative (-)

Statistics:

no data

Species / strain:

mammalian cell line, other: CHL/IU cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

none

-main test, pulse treatment: The incidence of cells with structurally and numerically aberrant chromosomes was less than 5%

-continuous treatment: The incidence of cells with structurally and numerically aberrant chromosomes was less than 5%

Conclusions:

The test material was considered to be not genotoxic

Executive summary:

A study was conducted to determine the potential genotoxicity of the test material using the in vitro chromosomal aberrations test.

Test substance was judged not to have the potential to induce chromosomal abberation since the incidence of cells with structurally and numerically aberrant chromosomes was less than 5%

The test material was considered to be not-genotoxic under the conditions of this test.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

weight of evidence

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

5000, 2500, 1250, 625 µg/mL

Vehicle / solvent:

distilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

EMS and CP

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Chromosome aberrations:

METHOD OF APPLICATION: in suspension

DURATION
- Preincubation period: 3 days
- Exposure duration: 20h (assay 1) and 28h (assay2)

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): 5% Giemsa solution

NUMBER OF REPLICATIONS: two (per concentration and per assay)

NUMBER OF CELLS EVALUATED: at least 200 well-spread metaphase cells for each concentration (per assay). The examination of slides from a culture was halted when 15 or more metaphases with aberrations (excluding gaps) have been recorded for that culture

Evaluation criteria:

The test item is considered to have shown clastogenic activity in this study if all of the following criteria are met:
-Increases in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (only data without gaps will be considered).
-The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
-The increases are statistically significant.
-The increases are not associated with large changes in pH or osmolarity of the treated cultures.

The historical control data for this laboratory were also considered in the evaluation. Evidence of a dose-response relationship (if any) was considered to support the conclusion.

The test item is concluded to have given a negative response if no reproducible, statistically significant increases are observed.

Statistics:

Fisher’s exact test

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: +S9: no, but tested up to precipitating conc; -S9: yes

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Remarks:

valid historical negative controls database

Positive controls validity:

valid

Conclusions:

Chinese hamster V79 cells treated with indium trihydroxide up to 5000 µg/mL did not show any consistent genotoxic activity under the conditions of this study

Executive summary:

The test item Indium trihydroxide was tested for potential clastogenic activity using the Chromosome Aberration Assay. The study included two Concentration Selection Cytotoxicity Assays and two Chromosome Aberration Assays.

 

The performed experiments were considered to be valid and to reflect the real potential of the test item to cause structural chromosomal aberrations in the cultured V79 Chinese hamster cells used in this study.

 

The effect of the test item on the frequency of the cells with structural chromosome aberrations without gaps was investigated either in the presence or absence of a metabolic activation system which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/b-naphthoflavone induced rats. Although, statistically significantincrease was seen in the number of cells with structural chromosome aberrations in one case in the experiment with metabolic activation of Assay 1, it was not repeatable, and there was no evidence of any dose response.

 

Although there was an isolated positive response in only one of two replicates with metabolic activation (3h treatment, 20h harvest), the increase was not repeatable and there was no dose response. Hence it is concluded that this individual increase does not represent a positive effect of the test item.

In conclusion, Chinese hamster V79 cells treated with indium trihydroxide up to 5000 µg/mL did not show any consistent genotoxic activity under the conditions of this study.

 

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

5000; 1666.7; 555.6; 185.2; 61.73 and 20.58 µg/mL.

Vehicle / solvent:

distilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

4-Nitroquinoline-N-oxide (NQO) and Cyclophosphamide monohydrate (CP)

Positive control substance:

4-nitroquinoline-N-oxide

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: When cells were growing well, subcultures were established in an appropriate number of flasks (after thawing, the cells were subcultured no more than 5 times before used in the study).
- Exposure duration: In Assay 1, cells were treated for 3-hours in the presence and absence of S9 mix. In Assay 2, cells were treated for 3-hours in the presence of S9 mix and for 24-hours in the absence of S9 mix.

SELECTION AGENT (mutation assays): TFT (5-trifluorothymidine)

NUMBER OF REPLICATIONS: two per treatment

DETERMINATION OF CYTOTOXICITY
- Method: plating efficiency, Suspension Growth (SG) and Relative Total Growth (RTG)

Evaluation criteria:

The test item was considered to be mutagenic in this assay if all the following criteria were met :
1. The assay is valid.
2. Statistically significant (p < 0.05) and biologically relevant increases in mutation frequency are observed in treated cultures compared to the corresponding negative (vehicle) control values at one or more concentrations.
3. The increases in mutation frequency are reproducible between replicate cultures and/or between tests (under the same treatment conditions).
4. There is a significant concentration-relationship as indicated by the linear trend analysis (p < 0.05).
5. The mutation frequency at the test concentration showing the largest increase is at least 126 mutants per 106 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative (vehicle) control value.

Statistics:

Dunnett’s test for multiple comparisons. T-test for independent samples.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Remarks:

valid historical negative controls database

Positive controls validity:

valid

The test item was examined up to 5000 µg/mL in the Preliminary Toxicity Test. Based on the results of the preliminary experiment, the following test item concentrations were examined in the mutation assays: 5000; 1666.7; 555.6; 185.2; 61.73 and 20.58 µg/mL.

In Assays 1-2, insolubility was detected in the final treatment medium at the end of the treatment in the 5000-185.2 µg/mL concentration range in the experiment with and without metabolic activation. There were no large changes in pH or osmolality after treatment.

In Assay 1, following a 3-hour treatment with metabolic activation, no cytotoxicity of the test item was observed. Therefore, an evaluation was made using data of all the six examined concentrations. Nosignificant increase in the mutation frequency was observed at the evaluated concentrations. Nodose response to the treatment was indicated by the linear trend analysis.

In Assay 1, following a 3-hour treatment without metabolic activation, no cytotoxicity of the test item was observed. Therefore, an evaluation was made using data of all the six examined concentrations. No statistically significant increase in the mutation frequency was observed at the evaluated concentrations.Furthermore, no significant dose response to the treatment was indicated by the linear trend analysis. This assay was considered as negative.

In Assay 2, following a 3-hour treatment with metabolic activation, similarly to the first test, no cytotoxicity was observed. An evaluation was made using data of the six examined concentrations. No statistically significantincrease in the mutation frequency was observed at any of the evaluatedconcentrations.Nodose response to the treatment was indicated by the linear trend analysis.

In Assay 2, following a 24-hour treatment without metabolic activation, no cytotoxicity of the test item was observed. An evaluation was made using data of the six examined concentrations. No statistically significant increases in the mutation frequency were observed at the evaluated concentrations. A statistically significant increase in the mutation frequency was observed at 555.6 µg/mL concentration.

However, the difference between the mutation frequency of the test item treated sample and the corresponding vehicle control value did not exceed the global evaluation factor, thus it was considered as biologically non relevant increase. Furthermore, no dose response to the treatment was indicated by the linear trend analysis. Therefore, Assay 2 was considered as confirmed the negative results of the first main test.

The experiments were performed using appropriate untreated, negative (vehicle) and positive control samples in all cases. The spontaneous mutation frequency of the negative (vehicle) controls was in the recommended range in each test. The positive controls gave the anticipated increases in mutation frequency over the controls. The plating efficiencies for the vehicle controls at the end of the expression period were within the acceptable range in all assays. The evaluated concentration ranges were considered to be adequate. The number of test concentrations met the acceptance criteria. Therefore, the overall study was considered to be valid.

Conclusions:

No mutagenic effect of Indium trihydroxide was observed either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.

Executive summary:

The Mouse Lymphoma Assay with Indium trihydroxide on L5178Y TK^+/-3.7.2 C cells was considered to be valid and to reflect the real potential of the test item to cause mutations in the cultured mouse cells used in this study.

 

Treatment with the test item did not result in a statistically and biologically significant increase in mutation frequencies either in the presence or absence of a rat metabolic activation system (S9 fraction) in the Mouse Lymphoma Assay.

 

In conclusion, no mutagenic effect of Indium trihydroxide was observed either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In an OECD test Guideline 474 study, conducted to GLP, no induction of micronuclei in bone marrow erythrocytes was observed following oral gavage of indium hydroxide to mice at up to and including 2000mg/kg bw/day.

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

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

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

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

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

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.

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.

- 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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In the chromosome aberrations test in Chinese hamster lung CHL/IU cells, equivalent to OECD 473, indium did not induce chromosome aberrations in vitro, both in the absence and presence of metabolic activation (Asakura, 2009). The authors remarked that 'the average particle size of indium was smaller than the cell size used in the test because it was impossible to make smaller indium particles due to the feature of metallic indium'. Therefore, they suggest 'it might be difficult to interpret the results without identifying its absorption rate by the cells'.

To strengthen in a weight of evidence approach the negative results in the chromosome aberration test with indium, read across is performed from the chromosomal aberration test data with indium hydroxide.

Justification for classification or non-classification

Based on the negative results from the in vitro Ames test, Mouse lymphoma test and chromosome aberration test together with the negative results from the in vivo mouse micronucleus test, classification for mutagenicity is not required according to the EU CLP criteria (EU 1272/2008)