Graphene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 Feb 2020 to 22 Apr 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Deviations:

no

Remarks:

There were no deviations from the regulations that affected the overall integrity of the study or the interpretation of the study results and conclusions.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

carbon

EC Number:

801-282-5

Cas Number:

1034343-98-0

Molecular formula:

Cx

IUPAC Name:

carbon

Test material form:

solid: nanoform

Specific details on test material used for the study:

Test item was insoluble. Therefore, its ability to form a suitable suspension in a suspending agent known to be compatible with the test system was assessed in the preliminary cytotoxicity test. The test item was found to form a suitable homogeneous formulation in aqueous 0.1% (w/v) carboxymethylcellulose sodium such that the maximum concentration recommended by the guideline was readily achievable.

Method

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells:
Primary cultures of human peripheral blood lymphocytes
- Suitability of cells:
stable chromosome number and structure (karyotype); low and stable background rate of aberrations; human cells are generally the most relevant for risk assessment

For lymphocytes:
- Sex, age and number of blood donors:
peripheral blood from a young (approximately 18-35 years of age), non-smoking, male donors
- Whether whole blood or separated lymphocytes were used:
whole blood

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Complete RPMI 1640 medium with supplemented RPMI 1640 medium with the following filter-sterilized components: 10% (v/v) fetal bovine serum, 50 μg gentamycin per mL and 4 units heparin per mL. For culturing, the incubator is set to maintain 37°C with 5% CO2 in a humidified atmosphere.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9
: Phenobarbital/5,6-benzoflavone induced male Sprague Dawley rat liver fraction supplied by Moltox
- concentration or volume of S9 mix and S9 in the final culture medium: 1 mL S9 mix in 4 mL culture medium. The S9 mix contained 10% v/v S9 fraction and the following sterile cofactors: 8 mM MgCl2, 33 mM KCl, 100 mM sodium phosphate buffer pH 7.4, 5 mM glucose-6-phosphate and 4 mM NADP

Test concentrations with justification for top dose:

0, 20.0, 63.2, 200, 632, 1000 µg/mL. The Preliminary Cytotoxicity Test found that the maximal concentration recommended by the guideline (2000 µg/mL) was readily achievable, however heavy precipitate/insoluble material was observed on the slides at 2000 μg/mL which was noted to interfere with the conduct of the test at the scoring level. Therefore, the maximum concentration had to be adjusted to 1000 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: aqueous 0.1% CMC (carboxymethylcellulose) sodium (w/v)
- Justification for choice of solvent/vehicle:
The suspending agent is known to be compatible with the test system. In a preliminary cytotoxicity test the test item was found to form a suitable homogeneous formulation in the suspending agent (aqueous 0.1% (w/v) CMC sodium).

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

aqueous 0.1% (w/v) CMC sodium

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

in sterile water

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicates
- Number of independent experiments : 1

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in dispersion.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: Whole blood was mixed with medium and phytohemagglutinin (to stimulate lymphocyte division) and placed in an incubator. Treatments were performed approximately 48 hours after culture initiation.
- Exposure duration/duration of treatment: 4-hour treatment period in the absence and presence of S9 mix and a 21-hour treatment period in the absence of S9 mix (after 4 hours medium change with complete medium)
- Harvest time after the end of treatment (sampling/recovery times): Cultures were harvested after specified duration of treatment by centrifugation.

FOR CHROMOSOME ABERRATION:
- Spindle inhibitor (cytogenetic assays): colcemid (0.1 μg/mL) was added 2 hours prior to harvesting
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The supernatant was discarded and the cell pellet was resuspended in 0.075 M KCl (hypotonic solution) and incubated in a water bath set to maintain 37°C for approximately 12 minutes. Fixative (3:1 methanol:acetic acid, v/v) was mixed with the cells suspended in hypotonic solution to reach a final concentration of approximately 20% v/v. Following another centrifugation, the supernatant was discarded and the cells were treated with three changes of fixative. After the third change of fixative, the cell pellet was collected by centrifugation and resuspended in fixative at an appropriate density for slide preparation. The fixed cells were dropped onto clean slides and air-dried before staining. At least two slides were prepared from each culture. The slides were washed with 3 changes of purified water (in-house), for at least 1 minute per change. The washed slides were stained with 10% v/v Giemsa stain for 15 minutes, washed in one change of purified water (prepared in-house) for at least 1 minute, washed in running tap water for no longer than 30 seconds, air-dried then mounted with coverslips using synthetic mountant.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): A total of 300 cells/readable metaphases per experimental point were examined for the presence of chromosome aberrations
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Readable metaphases are identified by the following criteria: i) chromosome number between 44 and 48 in a single stage of condensation, ii) well-spread with minimal overlap of chromosomes and chromosome arms, iii) chromatids separate with centromere intact, iiii) structure of chromosomes clear and well-defined. The International System for Human Cytogenetic Nomenclature (2013) was followed to designate the observed aberrations.
- Determination of endoreplication: yes
- Determination of polyploidy: yes

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index (MI); relative mitotic index (RMI)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Cells were examined for the presence of chromosome aberrations by Light microscopy using oil-immersion optics.

Rationale for test conditions:

The highest test item concentration tested in the preliminary cytotoxicity test was the maximum concentration recommended by the OECD TG (2000 μg/mL). Based on mitotic index results, no limiting cytotoxicity was noted up to 2000 μg/mL for any of the treatment regimes. However, at the highest concentration (2000 μg/mL), heavy precipitate/insoluble material was observed on the slides and was noted to interfere with the conduct of the test, at the scoring level. As per the OECD guideline, at the concentration producing a precipitate, care should be taken to assure that the precipitate does not interfere with the conduct of the test (e.g. staining or scoring). Therefore, the high concentration evaluated in the main test was 1000 μg/mL, the potential maximum feasible concentration, which was confirmed to not interfere with scoring.

In the absence of overt toxicity, the highest concentration of the test item selected for detailed analysis of chromosome aberrations for each treatment regime in the main test was the highest concentration tested (1000 μg/mL). In addition, all lower concentrations were also subjected to examination due to the observed differences in the formation of aggregates (i.e. insoluble material/precipitation).

Evaluation criteria:

CLEARLY POSITIVE (evidence of genotoxicity):
- At least one of the test item concentrations selected for detailed chromosome analysis exhibit a statistically significant increase in the incidence of aberrant cells compared with the concurrent negative control (p ≤ 0.05) at a concentration that does not greatly exceed a 55% reduction in mitotic index
- The increase is concentration-related when evaluated with an appropriate trend test (a trend test will be performed when the incidence of aberrant metaphases falls outside the distribution of the historical negative control database (e.g. 95% control limits)).
- The increase is outside the distribution of the historical negative control database (e.g. 95% control limits).
- If no statistical analysis is performed (e.g. when number of metaphases to be assessed has been reduced due to a high number of aberrant metaphases observed). The test item will be considered clearly positive if at least two of the test item concentrations selected for detailed chromosome analysis exhibit a substantial concentration-related increase (over the 95% control limits) in the incidence of aberrant cells compared with the concurrent negative control at a concentration that does not greatly exceed a 55% reduction in mitotic index.

CLEARLY NEGATIVE (no evidence of genotoxicity):
- None of the tested concentrations selected for detailed chromosome analysis exhibit a statistically significant increase (p > 0.05) in the incidence of aberrant cells compared to the concurrent negative control.
- All results are inside the distribution of the historical negative control data (e.g. 95% control limits).
- Evidence supportive of exposure of, or toxicity to, the cells has been demonstrated.

An equivocal result is concluded if no definite judgment can be made to fit the above criteria.

Statistics:

- The results obtained for each treatment group were compared with the results obtained for the concurrent negative control group from the same treatment regime using the Fisher’s Exact Test.
- For the statistical analysis, results from replicate cultures were combined to facilitate interpretation and maximize the power of statistical analysis.
- As none of the test item-treated groups showed an incidence of aberrant cells outside the distribution of the historical negative control database, the one-sided Cochran-Armitage test, used to assess significance, at the 5% level, of a concentration-related increase in the incidence of aberrant cells across all considered groups, was not required.

Results and discussion

Test results

Species / strain:

lymphocytes: human

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:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility:
The test item was insoluble. The test item was found to form a suitable homogeneous formulation in aqueous 0.1% (w/v) carboxymethylcellulose sodium. Formulations were kept stirring during preparation and dosing.
- Precipitation and time of the determination: Insoluble material/precipitation was observed (by eye) in the culture medium at the end of treatment, in all treatment durations, starting at test item concentrations of 63.2 μg/mL.
- Other confounding effects:
No change in culture medium color was observed at any experimental point.

STUDY RESULTS
- Concurrent vehicle negative and positive control data
: The negative control results were within the 95% control limits of the distribution of the negative control database. The positive controls (MMC, CP) produced statistically significant increases in the number of aberrant cells compared with the concurrent negative controls. The values from the positive controls were also compatible with those generated in the historical positive control database, confirming the sensitivity of the test system and the effectiveness of the S9 mix.

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o For lymphocytes in primary cultures: Based on mitotic index results, no limiting cytotoxicity was noted up to 2000 μg/mL for any of the treatment regimes.
- Genotoxicity results
o Number of cells scored for each culture and concentration, number of cells with chromosomal aberrations and type given separately for each treated and control culture, including and excluding gaps:
The test item did not cause any statistically significant increases in the proportion of aberrant metaphases at any experimental point.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data:

Cyclophosphamide, 4 hours in the presence of S9: range: 4.0 – 24.7; mean % aberrant cells: 12.7+- 4.0 (SD); no of treatments: 51
Mitomycin C, 21 hours in the absence of S9: range: 2.7 – 15.3; mean % aberrant cells: 9.1+- 2.6 (SD); no of treatments: 50
- Negative (solvent/vehicle) historical control data:
4 hours in the absence of S9: range: 0.00 – 1.70; mean % aberrant cells: 0.45 +- 0.38; no of treatments: 53
4 hours in the presence of S9: range: 0.00 – 2.70; mean % aberrant cells: 0.55 +- 0.56; no of treatments: 61
21 hours in the absence of S9: range: 0.00 – 2.70; mean % aberrant cells: 0.68 +- 0.60; no of treatments: 58

Any other information on results incl. tables

Cellular Uptake Assessment by Electron Microscopy:

Assessment of the treated samples and resulting TEM images found no definitive evidence of nanomaterial uptake (or cell membrane association) in the cultured lymphocytes. However, the assessment included a very small population of cells relative to the entire pellet, thus a low rate of cellular uptake cannot be excluded based on this study.

Applicant's summary and conclusion

Conclusions:

It is concluded that the test item did not show any evidence of genotoxic activity in this in vitro test for induction of chromosome damage, under the conditions tested. TEM found no clear evidence for cellular uptake of the test item in human lymphocytes after 21 hours exposure, and no cytotoxicity was observed up to 2000 μg/mL, indicating very limited capacity of the test item for cellular uptake, if any. However, irrespective of the question of a potentially minor extent of cellular internalization, this study in human lymphocytes demonstrates that low quantities of the test item potentially entering the cells or excess quantities surrounding the cells clearly do not directly or indirectly induce any chromosomal damage.

Executive summary:

Study design

This fully reliable GLP-study was performed according to OECD TG 473. The objective of this study was to determine the potential genotoxicity of Graphene (nano platelet) using an in vitro mammalian chromosome aberration test in human peripheral blood lymphocytes.

 

Results

A preliminary cytotoxicity test was used to determine concentrations for the main test. Human peripheral blood lymphocytes were exposed to the test item, prepared in aqueous 0.1% (w/v) carboxymethylcellulose sodium, at final concentrations ranging from 6.32 to 2000 μg/mL for 4 hours, in absence or presence of metabolic activation (S9), and 21 hours in absence of S9. No limiting cytotoxicity was noted at any concentration. However, at the highest concentration (2000 μg/mL), heavy precipitate/insoluble material was observed on the slides and was noted to interfere with the conduct of the test, at the scoring level. Therefore, in the main test, 1000 μg/mL was evaluated and confirmed as the maximum feasible concentration.

Human peripheral blood lymphocytes were treated as detailed above, harvested and metaphase preparations were assessed for chromosome aberrations. Analysis by transmission electron microscopy (TEM) of satellite cultures prepared during the study did not find cellular uptake of the test item at any of the concentrations tested. However, the assessment included a very small population of cells relative to the entire pellet and may depict the situation of the dynamic interplay between potential uptake and release at one time point only. Nevertheless, taking the observed formation of large aggregates upon contact with aqueous solutions going along with considerably large particle sizes into account, this finding indicates that it is highly likely that the test item seems not to be able to enter the cells. This is supported by the absence of any cytotoxicity observed in the preliminary cytotoxicity test.

The negative control incidences of aberrant metaphases were within the 95% control limits of the distribution of the negative control database. The response of the cultures to the positive controls (MMC and CP) confirmed the sensitivity of the test system and the activity of the S9 mix. Therefore, the results from both the negative and positive controls confirmed the validity of the assay.

No limiting cytotoxicity was observed at any of the test item concentrations evaluated. Cultures treated up to concentrations showing insoluble material/precipitate in the culture medium at the end of treatment did not show any statistically significant increases in the incidence of aberrant metaphases.

 

Conclusion

It is concluded that the test item did not show any evidence of genotoxic activity in this in vitro test for induction of chromosome damage, under the conditions tested. TEM found no clear evidence for cellular uptake of the test item in human lymphocytes after 21 hours exposure, and no cytotoxicity was observed up to 2000 μg/mL, indicating very limited capacity of the test item for cellular uptake, if any. However, irrespective of the question of a potentially minor extent of cellular internalization, this study in human lymphocytes demonstrates that low quantities of the test item potentially entering the cells or excess quantities surrounding the cells clearly do not directly or indirectly induce any chromosomal damage.