[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Jan - 04 Feb 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No 440/2008 of 30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM, UK GLP Monitoring Authority

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Vapour grown graphitic carbon fibre

Molecular formula:

C

IUPAC Name:

Vapour grown graphitic carbon fibre

Test material form:

solid

Method

Target gene:

his operon for S. typhimurium strains and trp operon for E. coli strain

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli, other: WP2uvrA-

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9-mix), prepared from livers treated with phenobarbitone/ß-naphthoflavone (80/100 mg/kg bw/day)

Test concentrations with justification for top dose:

Preliminary Toxicity Test:
S. typhimurium TA100 and E. coli WP2uvrA-: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation

Experiment 1 (range-finding test) and Experiment 2 (main test), plate incorporation method:
All strains: 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was insoluble in sterile distilled water, DMSO, acetone, dimethyl formamide and acetonitrile at 50 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks. The test material formed the best doseable suspension in DMSO, therefore, this solvent was selected as the vehicle.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-Aminoanthracene (2AA): +S9, 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537 and 10 µg/plate for WP2uvrA­ Benzo(a)pyrene (BP): +S9, 5 µg/plate for TA98

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION:
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: Inspection of the bacterial background lawn

Evaluation criteria:

Criteria for a positive result: A dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation.

Biological relevance of the results will be considered first. Statistical methods can also be used as an aid to evaluation. However, statistical significance will not be the only determining factor for a positive response.

A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Mean values and standard deviations were calculated.

Results and discussion

Test results

Key result

Species / strain:

other: S. typhimurium TA1535, TA1537, TA98 and TA100 and E. coli WP2uvrA-

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: no, but tested up to limit dose

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A black, powdery precipitate was observed at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%):
- Data are attached in separate pdf document. Refer to section 'Attached background material'.

Any other information on results incl. tables

Due to the huge quantity and complexity of the results, data are attached in a separate pdf document. Refer to section 'Attached background material'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative