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Genetic toxicity: in vitro

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in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-12-01 - 2018-03-01 (experimental phase)
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
OECD Guidelines for the Testing of Chemicals No. 476 “In Vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes“ (adopted 29 July 2016)
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material form:
solid: particulate/powder
Details on test material:
98.9 % Purity corrected for in formulation
Storage conditions: Room temperature in the dark
Specific details on test material used for the study:
- Source and lot/batch No.of test material: Batch No RW07_10


Target gene:
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Source of cells: Laboratory for Mutagenicity Testing; Techni-cal University, 64287 Darmstadt, Germany
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50%) both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22.
- Cell cycle length, doubling time or proliferation index: doubling time 12 - 16 h in stock cultures
- Modal number of chromosomes: 22
- Normal (negative control) cell cycle time: doubling time 12 - 16 h in stock cultures

- Type and identity of media including CO2 concentration if applicable: For seeding of the cell cultures the complete culture medium was MEM (Eagles Minimal Essential Medium) supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10% fetal bovine serum (FBS). During treatment no FBS was added to the medium. For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine.
- HAT medium used for cell cleansing: this is MEM growth medium supplemented with Hypoxanthine (13.6 µg/mL, 100 µM). Aminopterin (0.0178 µg/mL, 0.4 µM) and Thymidine (3.85 µg/mL, 16 µM)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Results from the preliminary cytotoxicity test were used to select the test item concentrations for the mutagenicity experiment.
The concentration range of test item was 0.03 to 1 µg/mL in both exposure groups: 0, 0.03, 0.06, 0.13, 0.25, 0.5 µg/mL without S9; 0, 0.03, 0.06, 0.13, 0.25 µg/mL with S9
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: MEM (Gibco)
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
Details on test system and experimental conditions:
- Cell density at seeding (if applicable): 1.0 E7 cells/225 cm2 flask were seeded

- Preincubation period: 24h
- Exposure duration: 4h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days

SELECTION AGENT (mutation assays): 11 μg/mL 6-thioguanine

NUMBER OF REPLICATIONS: duplicate cultures per concentration level

- Method: cloning efficiency
Rationale for test conditions:
as indicated by the guideline
Evaluation criteria:
Evaluation of Results
A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).
A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (based 95% control limits).
In cases when the response is neither clearly negative nor clearly positive as described above, or in order to judge the biological relevance of a result, the data should be evaluated by expert judgement or further investigations.
Statistical Analysis
When there is no indication of any marked increases in mutant frequency at any concentration then statistical analysis may not be necessary. In all other circumstances comparisons will be made between the appropriate vehicle control value and each individual concentration, using Student's t-test. Other statistical analysis may be used if they are considered to be appropriate.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
Untreated negative controls validity:
not applicable
Positive controls validity:
Additional information on results:
- Effects of pH: none, Solvent control: 7.48, 120 μg/mL test item: 7.45
- Effects of osmolality: Solvent control: 312 mOsm, 120 μg/mL test item: 309 mOsmnone

The pre-experiment was performed in the presence and absence (4 h treatment) of metabolic activation. Test item concentrations between 0.002 μg/mL and 0.5 μg/mL were used. The dose levels were selected to avoid excessive precipitation as indicated in the solubility test.
At the end of the exposure period, precipitate of the test item was observed at 0.5 pg/mL in both exposure groups.
The results of the individual flask counts and their analysis are presented in Table 1. There was no evidence of concentration related reductions in cloning efficiency in either of the exposure groups.
The maximum concentration selected for the main mutagenicity experiment was therefore limited by the onset of precipitate in both the absence and presence of metabolic activation.

Applicant's summary and conclusion

The test item, Graphene platelet (nano) did not induce any statistically significant or concentration-related increases in mutant frequency per survivor in either the absence or presence of metabolic activation. The test item was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.
Executive summary:

The study was performed according to OECD TG 476 under GLP to investigate the potential of Graphene platelet (nano) to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The treatment period was 4 hours with and without metabolic activation.

The concentrations used in the main test were selected using data from the preliminary toxicity test at a concentration range of 0.002 to 0.5 µg/mL. The maximum dose levels were limited by precipitate. The concentrations of test item plated for cloning efficiency and expression of mutant colonies were as follows: 0, 0.03, 0.06, 0.13, 0.25 and 0.5 µg/mL

No substantial and dose dependent increase of the mutation frequency was observed in the main experiment.

Appropriate reference mutagens, used as positive controls, induced marked increase in mutant frequencies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, Graphene platelet (nano) is considered to be non-mutagenic in this HPRT assay.