Graphite, acid-treated

Genetic toxicity: in vitro

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:

29 October 2010 - 16 March 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was generated according to internationally accepted guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP according to Chemikaliengesetz and Directive 2004/9/EC

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Graphite, acid-treated

EC Number:

291-367-8

EC Name:

Graphite, acid-treated

Cas Number:

90387-90-9

Molecular formula:

C(Carbon) + acid + oxidant Not available by EC, (CnX mHX)

IUPAC Name:

carbon

Details on test material:

- Name of test material (as cited in study report): graphite, acid treated
- Substance type: inorganic
- Physical state: soild
- Lot/batch No.: 1706
- Expiration date of the lot/batch: not applicable
- Storage condition of test material: at room temperature
- Colour: grey
- Density: 2.2 g/cm³
- Particle size range: 80% < 50 mesh

Method

Target gene:

Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102.

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: TA 98: his D 3052; rfa-; uvrB-; R-factor: frame shift mutations TA 100: his G 46; rfa-; uvrB-; R-factor: base-pair substitutions TA 1535: his G 46; rfa-; uvrB-: base-pair substitutions TA 1537: his C 3076; rfa-; uvrB-:

Metabolic activation:

with and without

Metabolic activation system:

2-AA; 2-aminoanthracene

Test concentrations with justification for top dose:

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.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 0.9% NaCl-solution (as polar extraction medium); DMSO (non-polar extraction medium).
- Weight/volume ratio: 0.2 g/mL according to ISO 10993-3, 2003 and ISO 10993-12, 2007
- Justification for choice of solvent/vehicle: stability of test substance in polar and non-polar extraction medium

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

extraction medium and A. dest

Controls 2

Positive controls:

yes

Remarks:

S. typhimurium: TA 100, TA 1535; without metabolic activation

Positive control substance:

sodium azide

Remarks:

Dissolved in: Aqua dest. Concentration: 10 µg /plate

Controls 3

Positive controls:

yes

Remarks:

S. typhimurium: TA 98, TA 1537; without metabolic activation

Positive control substance:

other: 4-NOPD; 4-nitro-o-phenylene-diamine

Remarks:

Dissolved in: DMSO Concentrations: 10 µg/plate for TA 98 40 µg/plate for TA 1537

Controls 4

Positive controls:

yes

Remarks:

S. typhimurium: TA 102; without metabolic activation

Positive control substance:

methylmethanesulfonate

Remarks:

Dissolved in: Aqua dest. Concentration: 1 µL/plate

Controls 5

Positive controls:

yes

Remarks:

S. typhimurium: TA 98, TA 100, TA 1535, TA 1537 and TA 102; with metabolic activation

Positive control substance:

other: 2-AA; 2-aminoanthracene

Remarks:

Dissolved in: DMSO Concentrations: 2.5 µg/plate ; 10 µg/plate for TA 102

Details on test system and experimental conditions:

PREPARATION OF THE TEST ITEM:
The test item was extracted in a polar extraction medium [0.9% NaCl Lot No.: Delta Select / 251002T-1 (physiological solution)] and in a non-polar extraction medium (DMSO Lot No.: Applichem / 9U009953) for 72 (+/- 2) h at 37 (+/- 1) °C at a weight/volume ratio of 0.2 g/mL according to ISO 10993-3, 2003 and ISO 10993-12, 2007. The extraction procedure did not reveal any abnormalities in the extraction medium or the test item. After extraction with the non-polar extraction medium DMSO a greyish colouring of the extract was observed. After extraction the polar and non-polar extracts were centrifuged at room temperature for 5 minutes at 1000 g. The polar extract was further processed by sterile filtration (filter manufacturer: Whatman, batch: D102229, material: cellulose acetate, pore size: 0.2 μm).

TEST SYSTEM
BACTERIA:
Five strains of S. typhimurium with the following characteristics were used:
TA 98:
his D 3052; rfa-; uvrB-; R-factor: frame shift mutations

TA 100:
his G 46; rfa-; uvrB-; R-factor: base-pair substitutions

TA 1535:
his G 46; rfa-; uvrB-: base-pair substitutions

TA 1537:
his C 3076; rfa-; uvrB-: frame shift mutations

TA 102:
his G 428 (pAQ1); rfa-; R-factor: base-pair substitutions

PREPARATION OF BACTERIA:
Samples of each tester strain were grown by culturing for 12 h at 38.5 °C in nutrient broth to the late exponential or early stationary phase of growth (approx. 109 cells/mL). The nutrient medium consists per litre:
8 g Nutrient Broth
5 g NaCl
A solution of ampicillin (125 µL, 10 mg/mL) (TA 98, TA 100 and TA 102) was added in order to retain the phenotypic characteristics of the strain.

AGAR PLATES:
The Vogel-Bonner Medium E agar plates with 2 % glucose used in the Ames Test were prepared by BSL BIOSERVICE GmbH or provided by an appropriate supplier. Quality controls were performed.
Vogel-Bonner-salts contain per litre:
10 g MgSO4 x 7 H2O
100 g citric acid
175 g NaNH4HPO4 x 4 H2O
500 g K2HPO4

Sterilisation was performed at 121 °C in an autoclave.

Vogel-Bonner Medium E agar plates contain per litre:
15 g Agar Agar
20 mL Vogel-Bonner salts
50 mL glucose-solvent (40%)
Sterilisation was performed at 121 °C in an autoclave.

OVERLAY AGAR:
The overlay agar contains per litre:
7.0 g Agar Agar
6.0 g NaCl
10.5 mg L-histidine x HCl x H2O
12.2 mg biotin
Sterilisation was performed at 121 °C in an autoclave.

S9 HOMOGENATE:
The S9 liver microsomal fraction was prepared at BSL BIOSERVICE GmbH. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route.
The following quality control determinations are performed:
a) Biological activity in:
- the Salmonella typhimurium assay using 2-aminoanthracene
- the mouse lymphoma assay using benzo[a]pyrene
- the chromosome aberration assay using cyclophosphamide.
b) Sterility Test:
A stock of the supernatant containing the microsomes was frozen in aliquots of 2 and 4.5 mL and stored at <= -75 °C.
The protein concentration in the S9 preparation (Lot: 230910) was 36 mg/mL. The S9 mix preparation was performed according to Ames et al.

PREPARATION OF S9 MIX:
The S9 mix preparation was performed according to Ames et al. 100 mM of ice cold sodium-ortho-phosphate-buffer, pH 7.4, was added to the following pre-weighed sterilised reagents to give final concentrations in the S9 mix of:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP
This solution was mixed with the liver 9000 x g supernatant fluid in the following proportion:
co-factor solution 9.5 parts
liver preparation 0.5 parts
During the experiment the S9 mix was stored on ice.

EXPOSURE CONCENTRATIONS:
The test item was tested with the following extract concentrations in the experiments:
10, 20, 40, 60, 80 and 100%

Evaluation criteria:

CRITERIA OF VALIDITY:

A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100, TA 102)
- the control plates with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range):

-S9 +S9
-------------------------
TA 98: 18 - 46 18 - 57
TA 100: 77 - 163 78 - 165
TA 1535: 5 - 29 5- 27
TA 1537: 5 - 30 5 - 36
TA 102: 164 - 390 163 - 472

- corresponding background growth on negative control, extract medium control and test plates is observed.
- the positive controls show a distinct enhancement of revertant rates over the control plate.


EVALUATION OF MUTAGENICITY:
The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the extract medium control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs

in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
than the reversion rate of the extract medium 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 is 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 results

Test results 1

Species / strain:

S. typhimurium TA 98

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

Test results 2

Species / strain:

S. typhimurium 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

Test results 3

Species / strain:

S. typhimurium TA 1535

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

Test results 4

Species / strain:

S. typhimurium TA 1537

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

Test results 5

Species / strain:

S. typhimurium TA 102

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

Any other information on results incl. tables

Extracts (polar and non-polar) of graphite, acid treated were investigated for their potential to induce gene mutations according to the pre-incubation method using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535, TA 1537 and TA 102.

Several concentrations of the test item extracts were used. Each assay was 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 observed 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. The reduction in the number of revertants down to a mutation factor of 0.4 found in tester strain TA 1535 at an extract concentration of 40% (with metabolic activation) and down to a mutation factor of 0.5 found in tester strain TA 1537 at an extract concentration of 60% (with metabolic activation), both found in experiment II, was regarded as not biologically relevant due to lack of a dose-response relationship.

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with extracts (polar and non-polar) of graphite, acid treated at any concentration level, neither in the presence nor absence of metabolic activation in both experiments.

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

A more comprehensive overview of the test results can be seen from the document attached below.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item extracts did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, extracts (polar and non-polar) of graphite, acid treated 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 graphite, acid treated for their ability to induce gene mutations the pre-incubation test was performed with theS almonella 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 extracts (polar and non-polar) of graphite, acid treated 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.