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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 December 2009 - 25 January 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study generated according to internationally accepted testing guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ISO 10993-1: 2009, "Evaluation and testing within a risk management process"
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ISO 10993-3: 2003, "Tests for genotoxicity, carcinogenicity and reproductive toxicity"
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Sulphuric acid, compound with graphite
EC Number:
235-819-4
EC Name:
Sulphuric acid, compound with graphite
Cas Number:
12777-87-6
Molecular formula:
Cm+ HSO4-* n H2SO4
IUPAC Name:
methane; sulfuric acid
Details on test material:
ECGA97914xH(21T9-2)

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
other: his D 3052; rfa-; uvrB-; R-factor
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
other: his G 46; rfa-; uvrB-; R-factor
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
other: his G 46; rfa-; uvrB-
Species / strain / cell type:
S. typhimurium TA 1537
Additional strain / cell type characteristics:
other: his C 3076; rfa-; uvrB-
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
other: his G 428 (pAQ1); rfa-; R-factor
Metabolic activation:
with and without
Test concentrations with justification for top dose:
The test item was tested with the following extract concentrations in the experiments: 10, 20, 40, 60, 80 and 100%.The 100% extract concentration corresponds to a weight/volume-ratio of 0.2 g/mL 9.9% NaCl resp. DMSO.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
untreated control
Untreated negative controls:
yes
Remarks:
vehicle alone
Positive controls:
yes
Remarks:
Tester strains: TA 100, TA 1535
Positive control substance:
sodium azide
Remarks:
concentration: 10 µg/plate; without metabolic activationMigrated to IUCLID6: purity: at least 99%; dissolved in aqua dest.
Positive controls:
yes
Remarks:
Tester strains: TA 98, TA 1537
Positive control substance:
other: 4-nitro-o-phenylene-diamine; purity: > 97%; dissolved in DMSO
Remarks:
concentrations: 10 µg/plate for TA 98, 40 µg/plate for TA 1537; without metabolic activation
Positive controls:
yes
Remarks:
Tester strain: TA 102
Remarks:
concentrations: 1 µL/plate; without metabolic activation
Positive controls:
yes
Remarks:
Tester strains: TA 98, TA 100, TA 1535, TA 1537, TA 102
Positive control substance:
other: 2-aminoanthracene; purity: 96%; dissolved in DMSO
Remarks:
concentrations: 2.5 µg/plate; 10 µg/plate for TA 102
Details on test system and experimental conditions:
For the pre-incubation method the following materials are mixed in a test tube and pre-incubated with the tester strains for 60 minutes at 37 °C: 100 µL Test extract at each dose level , vehicle control, negative control or reference mutagen solution (positive control), 500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),100 µL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain).After 60 min of pre-incubation, overlay agar (2000 µL) is added and poured onto the surface of a minimal agar plate. For each strain and dose level, including the controls, a minimum of three plates (in one case two plates) is used. After solidification the plates are inverted and incubated at 37 °C for at least 48 h in the dark.
Evaluation criteria:
Cytotoxicity was detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the vehicle control.A test was considered acceptable if for each strain:- the bacteria demonstrated their typical responses to ampicillin (TA 98, TA 100, TA 102)- the control plates without S9 mix were within the following ranges (mean values of the spontaneous reversion frequency within the historical control data range):-S9+S9TA 98:18 - 4618 – 61TA 100: 75 – 15981 – 165TA 1535:5 – 295 – 31TA 1537:5 – 305 – 36TA 102165 - 390163 - 541- corresponding background growth on negative control, vehicle control and test plates was observed- the positive controls showed a distinct enhancement of revertant rates over the control plateThe Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the vehicle control.A test item was considered as mutagenic if:- a clear and dose-related increase in the number of revertants occurred and/or- a biologically relevant positive response for at least one of the dose groups occurred in at least one tester strain with or without metabolic activation.A biologically increase was regarded as relevant:- if in tester strains TA 100 and TA 102 the number of reversions was at least twice as high- if in tester strains TA 98, TA 1535 and TA 1537 the number of reversions was at least three times higherthan the reversion rate of the vehicle control.A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups was considered to be non-mutagenic in this system.
Statistics:
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Additional information on results:
No toxic effects of the test item extracts were observed in any of the five tester strains used up to the highest extract concentrations evaluated (with and without metabolic activation).No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Expandable Natural Graphite at any concentration level, neither in the presence nor absence of metabolic activation.The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item extracts didnot cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.Therefore extracts (polar and non-polar) of Expandable Natural graphite are considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

In order to investigate the potential of extracts (polar and non-polar) of Expandable Natural Graphite for their ability to induce gene mutations the pre-incubation test was performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102.

The test item extracts were tested at several concentrations. The assays were conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item extracts were prepared and used in the experiments: 10, 20, 40, 60, 80 and 100%

The 100% extract concentration corresponds to a weight/volume-ratio of 0.2 g/mL 0.9% NaCl resp. DMSO.

No toxic effects of the test item extracts were noted in any of the five tester strains used up to the highest extract concentrations evaluated (with and without metabolic activation) in experiment I and II.

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Expandable Natural Graphite at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.