Germanium

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Germanium dioxide

EC Number:

215-180-8

EC Name:

Germanium dioxide

Cas Number:

1310-53-8

Molecular formula:

GeO2

IUPAC Name:

Germanium dioxide

Test material form:

solid: particulate/powder

Details on test material:

Product Name: Germanium Dioxide
CAS No.: 1310-53-8
Lot No./Batch No.: 1449
Colour / Form: White powder
Molecular formula: GeO2
Molecular weight: 104.59
Purity: 99.999%
Water Solubility: 4.5 g/L @ 25ºC
Storage: Room temperature (15-30°C), cool and dry place
Handling Precautions: As per MSDS (Appendix VIII)
Supplier: Teck Metals Ltd.

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

Method

Target gene:

not applicable

Species / strain

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

Controls

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).

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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

Applicant's summary and conclusion

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.