Germanium

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) germanium dioxide did not induce any mutations in Salmonella typhimurium strains (TA98, TA100, TA1535, TA1537) and Escherichia Coli strain WP2uvrA with and without metabolic activation (S9) (Arulnesan, 2012).

In an OECD test Guideline 487 study, conducted to GLP, germanium dioxide did not induce an increase in the frequency of micronucleated cells in the mouse cell line L5178Y TK^+/-

either in the presence or absence of a rat liver metabolic activation (S9) (Sire, 2017).

In an OECD Test Guideline 476 HPRT Assay performed on CHO K1 Chinese hamster ovarian cells, conducted to CLP, germanium dioxide did not result in a statistically and biologically significant dose-dependent increase in mutation frequencies either in the presence or absence of a rat metabolic activation system (S9) (Kovacs, 2018)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

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 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21st July, 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Teck Metals Ltd., batch n°: 1449
- Expiration date of the lot/batch: Not applicable
- Purity test date: 99.999 %
- Colour / Form: White powder


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:Room temperature (15 to 30ºC)
- Stability under test conditions:Stable under normal temperatures/pressures
- Solubility and stability of the test substance in the solvent/vehicle: Stable under normal temperatures/pressures
- Handling Precautions: Standard laboratory procedures

Target gene:

not applicable

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9. Rat S9 corresponds to the 9,000 x g fraction of liver homogenate from male Sprague-Dawley rats treated with Aroclor 1254

Test concentrations with justification for top dose:

0, 9.4, 19, 38, 75, 150 and 300 μg per plate for the S. typhimurium strains
0, 75, 150, 300, 600 and 1500 μg per plate for E. coli.
Once plated and at the end of the incubation period, precipitate was visible only at the highest concentration of 1500 μg per plate. Therefore at this concentration the test item was evaluated at the limit of solubility in the test system (OECD, 1997). For the S. typhimurium strains, Germanium dioxide was tested near the limit of toxicity (OECD,1997). For all strains and conditions, a minimum of 5 concentrations were analyzable for mutagenicity (OECD, 1997).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: It was determined that Germanium dioxide was limited in solubility in a number of solvents including water at concentrations required for testing. Thus water was selected as the vehicle for this study as it the solvent most compatible with the preincubation test

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

cyclophosphamide

methylmethanesulfonate

other: 2-aminoanthracene (2-AMA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; each in the presence and
absence of S9

DURATION
- Preincubation period: 20 minutes prior to adding the top agar and plating
- Exposure duration: All plates were incubated at 37 +/- 2°C for 48 to 72 hours

NUMBER OF REPLICATIONS: 3

SCORING OF PLATES: The number of revertant colonies per plate will then be manually counted. If scoring has to be delayed, the plates will be stored at 5  3oC. The background lawn will be evaluated for evidence of toxicity and test item precipitation.

Evaluation criteria:

- Negative result (no evidence of genotoxicity) is concluded if there is no substantial increase in the number of colonies per plate; i.e. the results do not exceed the upper 98 percentile limit of the historical solvent/negative control range. A negative result indicates that the test item is non-mutagenic in S. typhimurium or E. coli.
- Positive result will be considered positive when there is a significant increase in the number of colonies per plate in comparison to the concurrent negative control and a concentration-related increase over the exposure range tested. For the cases that there is historical data available, a positive result has to present an increase in the number of revertant colonies per plate in comparison to the historical data for at least one experimental condition. Biological relevance of the results will be considered first. A statistical method may be used as an aid in evaluating the test results but it will not be the only determining factor. A positive result indicates that the test item induces point mutations in S. typhimurium or E. coli.
- Equivocal result: If no definite judgment can be made to fit the above criteria, even after repeated experiments, then the result will be described as equivocal. An equivocal result indicates that a definitive result cannot be made by performing the bacterial reverse mutation assay under the conditions described in this protocol.

Statistics:

Statistical analysis was applied to the numbers suspected to be abnormally high or to have a dose-related increase in revertant counts. The colony counts were transformed (square root) to normalize the data prior to using the one-sided Dunnett’s test (Mahon, G.A.T., et al., 1989).

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Germanium dioxide was not mutagenic to S. typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli strain, WP2 uvrA, under the test conditions.

Executive summary:

Germanium dioxide was evaluated for its potential to induce point mutations in Salmonella typhimurium strains, TA98, TA100, TA1535, TA1537 and Escherichia coli strain WP2 uvrA. The experimental design followed the “OECD Guideline for Testing of Chemicals - 471, Bacterial Reverse Mutation Test” (OECD, 1997). In the plate incorporation experiment, both in the presence and absence of S9, the concentrations of Germanium dioxide investigated were 0, 9.4, 19, 38, 75, 150 and 300 μg per plate for the S. typhimurium strains and 0, 75, 150, 300, 600 and 1500 μg per plate for E. coli. Once plated and at the end of the incubation period, precipitate was visible only at the highest concentration of 1500 μg per plate. Therefore at this concentration the test item was evaluated at the limit of solubility in the test system (OECD, 1997). For the S. typhimurium strains, Germanium dioxide was tested near the limit of toxicity (OECD, 1997). For all strains and conditions, a minimum of 5 concentrations were analyzable for mutagenicity (OECD, 1997). For the plate incorporation test, with or without metabolic activation, Germanium dioxide did not produce any increases in revertants over the concurrent negative controls. In the preincubation experiment, both in the presence and absence of S9, the concentrations of Germanium dioxide investigated were identical to the plate incorporation test. Solubility and toxicity results were similar to those of the plate incorporation test. The preincubation test confirmed the negative results of the plate incorporation test. Germanium dioxide did not produce any statistically significant increases in revertants over the concurrent negative controls. The negative controls for each tester strain were within the historical negative control data. All concurrent positive controls induced at least a 2.9-fold increase in colony counts per plate when compared to the corresponding negative controls and were at levels similar to the historical positive control data. Thus, it was concluded that Germanium dioxide was not mutagenic to S. typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli strain, WP2 uvrA, under the test conditions.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 January 2017- 16 May 2017

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 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

26 September 2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ATCC (American Type Culture Collection, Manassas, USA), by the intermediate of Biovalley (Marne-La-Vallée, France).
- Suitability of cells: established cell line recommended by international regulations for in vitro mammalian cell gene mutation test and for in vitro micronucleus test
- Cell cycle length, doubling time or proliferation index: 10-12 hours

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL). This medium was supplemented by heat inactivated horse serum at 10% (v/v).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability:no
- Periodically 'cleansed' against high spontaneous background: no

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

The S9 mix consists of induced enzymatic systems contained in rat liver post-mitochondrial fraction (S9 fraction) and the cofactors necessary for their function (c).

Test concentrations with justification for top dose:

preliminary test : 0.02, 0.2, 1, 2, 5 and 10 mM.
Since the test item was found to be non-severely cytotoxic in the preliminary test, the highest dose level selected for the main experiment was 10 mM, according to the criteria specified in the international regulations.
main experiment: 0.313, 0.625, 1.25, 2.5, 5 and 10 mM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium (RPMI 1640)
- Justification for choice of solvent/vehicle: Solubility assays were performed

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: colchicine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
- Cell density at seeding (if applicable):

DURATION
- Preincubation period:
- Exposure duration:
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays):

SPINDLE INHIBITOR (cytogenetic assays):

STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS:

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:

NUMBER OF CELLS EVALUATED:

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):

- OTHER:

Evaluation criteria:

Evaluation of a positive response: a test item is considered to have clastogenic and/or aneugenic potential, if all the following criteria were met:
a dose-related increase in the frequency of micronucleated cells was demonstrated by a statistically significant trend test,
for at least one dose-level, the frequency of micronucleated cells of each replicate culture was above the corresponding vehicle historical range,
a statistically significant difference in comparison to the corresponding vehicle control was obtained at one or more dose-levels.
Evaluation of a negative response: a test item is considered clearly negative if none of the criteria for a positive response was met.

Statistics:

For each condition of the cytogenetic experiment, the frequency of micronucleated cells in treated cultures was compared to that of the vehicle control cultures.
This comparison was performed using the chi2 test, unless treated culture data are lower than or equal to the vehicle control data. P = 0.05 was used as the lowest level of significance. This statistical analysis was performed using a validated Excel sheet.
To assess the dose-response trend, a linear regression was performed between the frequencies of micronucleated cells and the dose-levels. This statistical analysis was performed using SAS Enterprise Guide software.

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 applicable

Positive controls validity:

valid

Conclusions:

Under the experimental conditions of the study, the test item, Germanium dioxide, did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.

Executive summary:

The objective of this study was to evaluate the potential of the test item, Germanium dioxide, to induce an increase in the frequency of micronucleated cells in the mouse cell line L5178Y TK^+/-.

 

Methods

 

After a preliminary toxicity test, the test item Germanium dioxide, suspended in culture medium (RPMI 1640), was tested in a single experiment, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, as follows:

 

Without S9 mix	3 h treatment + 24 h recovery
	24 h treatment + 0 h recovery
With S9 mix	3 h treatment + 24 h recovery

 

Each treatment was coupled to an assessment of cytotoxicity at the at the same dose-levels. Cytoxicity was evaluated by determining the PD (population doubling) of cells

After the final cell counting, the cells were washed and fixed. Then, cells from at least three dose-levels of the test item-treated cultures were dropped onto clean glass slides. The slides were air-dried before being stained in 5% Giemsa. Slides from vehicle and positive controls cultures were also prepared. All slides were coded before analysis, so that the analyst was unaware of the treatment details of the slide under evaluation ("blind" scoring). For main experiment (with or without S9 mix), micronuclei were analyzed for three dose‑levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose).

Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture.

 

Results

Since the test item was found to be non-severely cytotoxic in the preliminary test, the highest dose‑level selected for the main experiment was 10 mM, according to the criteria specified in the international regulations.

 

The mean population doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Also, positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid.

 

Experiment without S9 mix

With a treatment volume of 1% (v/v) in the culture medium, the selected dose-levels were 0.313, 0.625, 1.25, 2.5, 5 and 10 mM for the 3- and 24-hour treatments.

 

No precipitate was observed in the culture medium at any dose-levels at the end of the treatment periods.

 

Cytotoxicity

Following the 3-hour treatment, no cytotoxicity was induced at any dose-levels, as shown by the absence of noteworthy decrease in the PD.

Following the 24-hour treatment, a moderate to severe cytotoxicity was induced at dose-levels ≥ 1.25 mM, as shown by a 45 to 81% decrease in the PD.

Micronucleus analysis

The dose-levels selected for the micronucleus analysis were as follows:

- 2.5, 5 and 10 mM for the 3-hour treatment, the latter being the highest recommended dose-level,

- 0.625, 1.25 and 2.5 mM for the 24-hour treatment, the latter inducing a 55% decrease in the PD

Following the 3-hour treatment, a slight but statistically significant increase in the frequency of micronucleated cells was observed at the dose-level of 5 mM relative to the vehicle control (3‰vs.0.0‰ for the vehicle control; p<0.05). No dose-response relationship was demonstrated by the linear regression (p>0.05) and all frequencies remained within the vehicle control historical range [0.0 - 3.5‰].

The slight statistical significance observed was considered to be only due to the very low frequency obtained in the vehicle control culture.

 

Following the 24-hour treatment, no statistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed dose-levels relative to the vehicle control (up to 5‰vs.3‰ for the vehicle control; p>0.05). A slight but statistically significant dose-response relationship was observed (demonstrated by the linear regression (p<0.05)). Since all frequencies remained within the vehicle control historical range [1 - 6‰] and in the absence of statistically significant difference with the vehicle control, the statistically significant linear trend was considered as meaningless in terms of genotoxicity.

 

The results of this experiment without S9 mix were considered to show a negative response.

 

Experiment with S9 mix

With a treatment volume of 1% (v/v) in the culture medium, the selected dose-levels were 0.313, 0.625, 1.25, 2.5, 5 and 10 mM.

 

No precipitate was observed in the culture medium at any dose-levels at the end of the treatment periods.

 

Cytotoxicity

No cytotoxicity was induced at any dose-levels, as shown by the absence of any noteworthy decrease in the PD.

 

Micronucleus analysis

The dose-levels selected for micronucleus analysis were 2.5, 5 and 10 mM, the latter being the highest recommended dose-level.

 

No increase in the frequency of micronucleated cells was observed at any of the analyzed dose-levels relative to the vehicle control (up to 2.5‰vs.2.5‰ for the vehicle control; p>0.05).

No dose-response relationship was demonstrated by the linear regression (p>0.05) and all frequencies remained within the vehicle control historical range [0.0 - 3.5‰].

These results met the criteria of a negative response.

 

Conclusion

 

Under the experimental conditions of the study, the test item, Germanium dioxide, did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK^+/-mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.

 

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

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)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: mammalian cell gene mutation assay

Target gene:

Hprt gene

Species / strain / cell type:

mammalian cell line, other: Sub-line (K1) of Chinese hamster ovary cell line CHO

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

main mutation assay: 2000; 1000; 500; 250; 125; 62.5 µg/mL
Treatment concentrations for the mutation assays were selected on the basis of results of the performed Preliminary Toxicity Test according (concentrations tested: 2000; 1000; 500; 250; 125; 62.5; 31.25; 15.625; 7.813; 3.906) to the OECD guideline instructions (up to the maximum recommended concentration)

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

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
- Exposure duration: In Assay 1, cells were treated for 5-hours in the presence and absence of S9 mix. In Assay 2, cells were treated for 5-hours in the presence of S9 mix and for 24-hours in the absence of S9 mix.

SELECTION AGENT (mutation assays): 6-thioguanine (6-TG)

NUMBER OF REPLICATIONS: two per treatment

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, mutation frequency

Evaluation criteria:

The test item was considered to be mutagenic in this assay if the following criteria are met:
1. The assay is valid.
2. The mutant frequency at one or more doses is significantly greater than that of the relevant negative (vehicle) control (p<0.05).
3. Increase of the mutant frequency is reproducible.
4. There is a dose-response relationship.

Results which only partially met the criteria were dealt with on a case-by-case basis (historical control data of untreated control samples was taken into consideration if necessary). Similarly, positive responses seen only at high levels of cytotoxicity required careful interpretation when assessing their biological significance. In cases with survival lower than 10%, extreme caution is taken in the interpretation.

Statistics:

The homogeneity of variance between groups was checked by Bartlett`s homogeneity of variance test.
Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was made.
If the obtained result were significant, Duncan’s Multiple Range test was used to assess the significance of inter-group differences.
Significant results with inter-group comparisons were further compared using Kruskal-Wallis and Mann-Whitney U-tests.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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

Conclusions:

The HPRT Assay with GERMANIUM DIOXIDE performed on CHO K1 Chinese hamster ovarian cells was considered to be valid and reflect the real potential of the test item to cause mutations in the cultured mammalian cells used in this study.

Treatment with the test item did not result in a statistically and biologically significant dose-dependent increase in mutation frequencies either in the presence or absence of a rat metabolic activation system (S9) in this study.

In conclusion, no mutagenic effect of GERMANIUM DIOXIDE was observed either in the presence or absence of metabolic activation system under the conditions of this HPRT assay.

Executive summary:

An in vitro mammalian cell assay was performed in CHO K1 Chinese hamster ovary cells at the hprt locus to evaluate the potential of GERMANIUM DIOXIDE to cause gene mutation. Treatments were carried out for 5 hours with and without metabolic activation (±S9-mix) and for 24 hours without metabolic activation (-S9-mix).

 

DMSO was used as the vehicle (solvent) of the test item in this study. Treatment concentrations for the mutation assays of the main tests were selected based on the results of a preliminary toxicity test as follows:

Assay 1

5-hour treatment in the presence of S9-mix:

2000, 1000, 500, 250, 125 and 62.5µg/mL

5-hour treatment in the absence of S9-mix:

2000, 1000, 500, 250, 125 and 62.5µg/mL

Assay 2

5-hour treatment in the presence of S9-mix:

2000, 1000, 500, 250, 125 and 62.5µg/mL

24-hour treatment in the absence of S9-mix:

2000, 1000, 500, 250, 125 and 62.5µg/mL

 

In the main assays, a measurement of the survival (colony-forming ability at the end of the treatment period) and viability (colony-forming ability at the end of the 7 day expression period following the treatment) and mutagenicity (colony forming ability at the end of the 7 day expression period following the treatment, in the presence of 6-thioguanine as a selective agent) was determined.

 

In Assay 1, insolubility (minimal amount of precipitate) was detected at 2000 µg/mL concentration in the final treatment medium at the end of the treatment in the experiments with metabolic activation. The precipitation did not interfere with the reading of the results. There were no large changes in pH and osmolality after treatment in any cases.

 

In Assay 2, no insolubility was detected in the final treatment medium at the end of the treatment with or without metabolic activation. There were no large changes in pH and osmolality after treatment in any cases.

 

In Assay 1 and 2, in the presence of S9-mix (5-hour treatment), no marked cytotoxicity of the test item was observed. An evaluation was made using data of all six concentrations. No statisticallysignificant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and therewas no dose response to the treatment (a trend analysis showed no effect of treatment).

 

In Assay 1, in the absence of S9-mix (5-hour treatment), no marked cytotoxicity of the test item was observed. An evaluation was made using data of all six concentrations. No statisticallysignificant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and therewas no dose response to the treatment (a trend analysis showed no effect of treatment)

 

In Assay 2, in the absence of S9-mix (24-hour treatment), no marked cytotoxicity of the test item was observed, thus an evaluation was made using data of all six concentrations. No statisticallysignificant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and therewas no dose response to the treatment (a trend analysis showed no effect of treatment). This experiment confirmed the negative results seen in Assay 1 without metabolic activation.

 

The spontaneous mutation frequency of the negative (vehicle) control was in accordance with the historical control range in all assays. The positive controls gave the anticipated increases in mutation frequency over the controls and were in good harmony with the historical data in all assays. Six evaluated concentrations were presented in all assays. The cloning efficiencies for the negative controls at the beginning and end of the expression period were within the target range. The evaluated concentration ranges were considered to be adequate (concentrations were tested up to the cytotoxic range in each test). The overall study was considered to be valid.

 

In conclusion, no mutagenic effect of GERMANIUM DIOXIDE was observed either in the presence or absence of a metabolic activation system under the conditions of this HPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on read across from the negative results from the in vitro Ames test, in vitro mammalian cell gene mutation (Hprt) test and in vitro micronucleus test with germanium dioxide, classification for mutagenicity is not required according to the EU CLP criteria (EU 1272/2008)