Graphene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Key, CA, OECD TG 473, GLP, with and without S9, 0, 20.0, 63.2, 200, 632, 1000 µg/mL: Negative (Registre, 2020)

Key, HPRT, OECD TG 467, GLP, with and without S9, 0.03 to 1 µg/mL: Negative (Brown, 2018)

Link to relevant study records

Reference

Reference 1

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

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

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.

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).

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.

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

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 Dec 2017 to 01 Mar 2018 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

hprt

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- 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

MEDIA USED
- 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:

yes

Negative solvent / vehicle controls:

yes

Remarks:

MEM

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 1.0 E7 cells/225 cm2 flask were seeded

DURATION
- 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

DETERMINATION OF CYTOTOXICITY
- 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.

Statistics:

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.

Species / strain:

Chinese hamster lung fibroblasts (V79)

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 applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- 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

RANGE-FINDING/SCREENING STUDIES:
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.

Conclusions:

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:

Study design

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

Results

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.

Conclusion

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Key, Combined In Vivo Mammalian Erythrocyte Micronucleus Test and Alkaline Comet Assay in Rat, OECD TG 474 and 489, GLP: no signs of genetic toxic effects (Merah, 2020)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Combined In Vivo Mammalian Erythrocyte Micronucleus Test and Alkaline Comet Assay in Rat

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Feb 2020 to 28 Apr 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes

Remarks:

The study was conducted according to GLP, except for characterization and stability of the test item, which was performed according to established SOPs, controls, and approved test methodologies to ensure integrity and validity of the results generated

Type of assay:

mammalian erythrocyte micronucleus test

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Canada Inc, St-Constant, QC, Canada.
- Age at study initiation: 49-50 days old
- Weight at study initiation: 181 to 216 (males) and 163 to 184 g (females)
- Assigned to test groups randomly: yes, under following basis: the weight variation of animals was confirmed to be minimal as it did not exceed ± 20% of the mean weight
- Housing: Animals were group housed (up to 3 animals of the same dosing group together) in polycarbonate cages containing appropriate bedding equipped with an automatic watering valve. These housing conditions were maintained for the duration of the study. Control group animals were housed on a separate rack from the test item-dosed animals.
- Diet (e.g. ad libitum): ad libitum throughout the study, except during designated procedures
- Water (e.g. ad libitum): Municipal tap water after treatment by reverse osmosis and ultraviolet irradiation was freely available to each animal via an automatic watering system (except during designated procedures).
- Acclimation period: A period of 7 to 8 days was allowed between animal receipt and the start of treatment in order to acclimate the animals to the laboratory environment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C to 25°C
- Humidity (%): 30% to 70%
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

Route of administration:

inhalation

Details on exposure:

TYPE OF INHALATION EXPOSURE: nose only

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Flow-through chamber, chamber type 4 with 2 levels per chamber and 40 breathing ports
- Method of holding animals in test chamber: Restraint tubes
- System of generating particulates/aerosols: Extended Duration Powder Delivery System with Jet Mill and Cycloneb
- Temperature, humidity, pressure in air chamber: 19-25°C, 30-70% relative humidity, and at least 19% O2

TEST ATMOSPHERE
- Brief description of analytical method used: Real-time Qualitative Monitoring: microdust; Aerosol Concentration Analysis, (Substrate/Diameter): Glass fiber/25 mm; Particle Size Analysis (Substrate/Diameter): Stainless steel plates/34 mm

Duration of treatment / exposure:

3 days at 0, 24 and 45 hours (±15 minutes), once daily for up to 240 minutes

Frequency of treatment:

3 days at 0, 24 and 45 hours (±15 minutes), once daily for up to 240 minutes

Dose / conc.:

0 mg/L air

Remarks:

Control

Dose / conc.:

0.5 mg/L air

Dose / conc.:

1 mg/L air

Dose / conc.:

2 mg/L air

Remarks:

according to the acute inhalation study, 2 mg/L air was the maximum respirable practical dose.

No. of animals per sex per dose:

5 per sex per dose

Control animals:

yes

Positive control(s):

Positive control: Cyclophosphamide (CP)
- Doses / concentrations: 20 mg/kg at 10 mL/kg given 24 hours prior to sampling

Tissues and cell types examined:

Both femurs (groups 1 to 4) were dissected from each main study animal. Several bone marrow cell smears were prepared from each animal. The bone marrow from both femurs of each animal was pooled/eluted in 5 mL Hanks Balance Salts Solution. The resulting cell suspensions were processed for the micronucleus test.

Microscopic examination: A total of 4000 immature erythrocytes per animal were examined for the presence of micronuclei.
In addition, the proportion of immature erythrocytes was assessed by examination of a total of at least 500 erythrocytes per animal. The incidence of any micronucleated mature erythrocytes observed during this assessment was recorded as a check for potential micronucleus-like artifacts.

Details of tissue and slide preparation:

PROCESSING
Several bone marrow cell smears were prepared from each animal.

DETAILS OF SLIDE PREPARATION
The slides were fixed in methanol and stained with the fluorescent metachromatic dye, acridine orange for micronucleus test evaluation.
Positive control slides taken from 3 Sprague Dawley male rats previously dosed with positive control (cyclophosphamide, 20 mg/kg at 10 mL/kg given 24 hours prior to sampling), as part of a CR-MTL GLP-compliant study, were added to the study slides for evaluation as scoring controls. Data from these animals were entered in the study as a positive control group called Group 6 for use in statistical evaluation. Arbitrary animal numbers were given based on the standard animal identification scheme. No slides from the positive control Group 6 were archived.

METHOD OF ANALYSIS
Microscopic Examination: The slides were encoded to minimize potential operator bias and then examined by fluorescence microscopy using a blue excitation filter and a yellow barrier filter. A total of 4000 immature erythrocytes per animal were examined for the presence of micronuclei. One smear was examined per animal, the remaining smears being held temporarily as reserves in case of technical problems with the first smear.

Evaluation criteria:

CRITERIA FOR BOTH COMET AND MICRONUCLEUS ASSAY

ACCEPTABILITY CRITERIA: concurrent positive and negative controls are within the distribution of the historical control data and positive control induce a statistically significant increase compared with the concurrent negative control; an appropriate number of doses and cells has been analysed (4000 PCE per animal for micronucleus test or 150 cells per tissue per animal for comet assay); The maximum dose tested is one that allows maximum exposure up to 2000 mg/kg/day for non-toxic compounds, or the limit of solubility, respirability or MTD based on available toxicity data, or the highest dose may also be defined as a dose that produces toxicity in the bone marrow.

CLEARLY POSITIVE
1. At least one of the test item groups exhibits a statistically significant increase in the frequency of MN-PCE or % tail DNA compared with the concurrent negative control.
2. The increase is dose-related when evaluated with an appropriate trend test.
3. Any of the results are outside the distribution of the historical negative control data (e.g. 95% control limits).
Conclusion: The test item is then considered able to induce DNA strand breakage/chromosomal damage or damage to the mitotic apparatus in the tissue studied in this test system.

CLEARLY NEGATIVE
1. None of the test item groups exhibits a statistically significant increase in the frequency of MN-PCE or % tail DNA compared with the concurrent negative control.
2. All results are inside the distribution of the historical negative control data (e.g. 95% control limits).
3. Direct or indirect evidence supportive of exposure of, or toxicity to, the target tissue(s) has been demonstrated.
Conclusion: The test item is then considered unable to induce DNA strand breakage/chromosomal damage or damage to the mitotic apparatus in the tissue studied in this test system.

Statistics:

All statistical tests were conducted at the 5% significance level. The pairwise comparisons were reported at the 0.1%, 1%, and/or 5% levels. Result was considered to be significant if p ≤ 0.05.

Numerical data collected on scheduled occasions were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation were reported whenever possible.
STATISTICS FOR BOTH COMET AND MICRONUCLEUS ASSAY

The numerical data corresponding to the MN-PCE frequency and % tail DNA was statistically evaluated using two separate datasets. The first one included the negative control group and the test item treated groups, whereas, the second one included only the negative control group and the positive control group.

For the first dataset, Levene’s test was used to assess the homogeneity of group variances. If the result of Levene’s test was not found to be significant (p > 0.05), then the groups were compared using an overall one-way ANOVA F-test, otherwise they were compared using Kruskal-Wallis test. If the overall ANOVA F-test or Kruskal-Wallis test was found to be significant (p ≤ 0.05),conducted using Dunnett’s or Dunn’s test, respectively.

For the second dataset, Levene’s test was used to compare the variances of negative and positive control groups. If the result of Levene’s test was not found to be significant (p > 0.05), then the groups were compared using t-test, otherwise, they were compared using Wilcoxon Rank-Sum
test.

Whenever the results of MN-PCE and % tail DNA, corresponding to test item treated groups, reveal significant mean increase when compared to the negative control group, then a doserelated increase trend in means across the negative control group and the test item treated groups would be conducted using Cochran-Armitage’s one-sided.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

There was no mortality, and no remarkable treatment-related clinical signs were noted during the study. Low body weight gain was observed at the highest respirable dose level (actual 1.92 mg/L) which is indicative of systemic toxicity.

Negative controls validity:

valid

Positive controls validity:

valid

Achieved chamber conditions:

Individual mass median aerodynamic diameter (MMAD) of generated aerosols ranged from 2.9 to 5.2 μm. Although this was higher than the targeted 4.0 μm aerosol, generated aerosols were considered acceptable given that the majority of particle size assessments described at least 30% of particles with a diameter < 3.5 μm. On the three consecutive days of inhalation dosing animals in Groups 1 to 4 were exposed to a stable and respirable aerosols of the test item at either 0, 0.547, 1.00 or 1.92 mg/L.

Results of In-life Procedures, Observations, and Measurements

- Mortality: No mortalities occurred during this study.

- Clinical Observations: There were no test item-related clinical signs observed during the dosing and observation period. Black fur staining appeared during the dosing period, and were no longer present at the end of dosing or remained present with slight severity 1 hour post the end of dosing. This observation can be attributed to the nose-only administration of the test item to the animals using a Flow-Through rodent system, and considered non-adverse because of their transient nature.

All other clinical observations were considered unrelated to the test item based on their low and/or sporadic incidence.

- Body Weights and Body Weight Gains: The body weight increased during the pre-treatment period for all the animals. During the treatment period, there was low body weight gain at 1.92 mg/L. The body weight gain values for Days -1 to 3 for the negative control group and test item dose level groups 0.547, 1.0, and 1.92 mg/L were for males: 37.0, 31.8, 35.4 and 23.2 g and for females: 19.8, 19.4, 17.6 and 16.8 g, respectively.

This low body weight gain is indicative of systemic toxicity.

Proportion of Polychromatic Erythrocytes

Animals treated with the test item did not show any notable decreases in %PCE/TE, indicating that graphene did not cause bone marrow toxicity following exposure by nose-only inhalation for 240 minutes for 3 days to male and female rats. Group mean values for %PCE/TE (51.7 to 61.4% for males and 49.3 to 54.7% for females) were within the historical negative control range of 27.2 to 72.9% and 28.7 to 62.9% for males and females, respectively.

Micronucleated Polychromatic Erythrocytes

Male and female rats treated with the test item did not show any statistically significant increases in the frequency of MN-PCE. The group mean values for negative and test item dose levels 0.547, 1.0, and 1.92 mg/L were 5.5, 3.4, 2.4 and 4.8 for males; and 4.0, 4.4, 5.2 and 4.2 for the females, respectively. These group mean values were below the 95% historical negative control upper limit (8.9 and 8.7 for males and females, respectively). Even though, a statistical significance was obtained in group 3 males, it is not considered biologically meaningful as the micronuclei frequency was even lower in this group than in the negative control group. Therefore, graphene did not induce chromosomal damage or damage to the mitotic apparatus as assessed by micronuclei formation in bone marrow polychromatic erythrocytes of male and female rats, when exposed by nose-only inhalation for 240 minutes for 3 days to male and female rats up to 1.92 mg/L which is considered the maximum respirable dose level. This dose level induced low body weight gain which is indicative of systemic toxicity.

Conclusions:

In conclusion, the graphene test material did not induce the formation of micronuclei in polychromatic erythrocytes in the micronucleus test, when administered by nose-only inhalation for 240 minutes for 3 days to male and female rats up to 1.92 mg/L, the maximum respirable practical dose. Also this dose level induced low body weight gain which is indicative of systemic toxicity.

Executive summary:

Study design

The objective of this GLP-study was to determine the potential genotoxicity of graphene when given by nose-only inhalation for 240 minutes for 3 days in rats using the bone marrow micronucleus test and the comet assay with the lung using OECD 474 and 489 guidelines.

In the study, rats were dosed using target concentrations of 0.5, 1, 2 mg/L of graphene (5 animals/sex/group). The dose levels were selected based on the results of acute inhalation toxicity study (Zabaiou, 2020), where it was determined that the test item at 2 mg/L is the maximum respirable practical dose. 20 mg/mL (10 mL/kg) Ethyl methanesulfonate (EMS) or cyclophosphamide was used as positive controls (3 male animals). Bone marrow and lung were collected 3 hours after the last treatment.

 

Results

The generated aerosols of graphene were considered to be within the respirable range for the rats and the target concentrations were achieved during the three consecutive days of dosing by inhalation. There was no mortality, and no remarkable treatment-related clinical signs were noted during the study. Low body weight gain was observed at the highest respirable dose level (actual 1.92 mg/L) which is indicative of systemic toxicity. A total of 4000 immature polychromatic erythrocytes (PCEs) were evaluated for presence of micronuclei indicative of chromosome damage. In addition, the proportion of immature erythrocytes in the total of at least 500 erythrocytes per animal was assessed as a measure of potential bone marrow toxicity. The proportion of polychromatic erythrocytes and the frequencies of micronucleated polychromatic erythrocytes (MN-PCE) in the negative control group were within the laboratory historical negative control range. The positive control, cyclophosphamide, induced a clear and unequivocal increase in micronuclei. The results from both the negative and positive controls confirmed the validity of the assay. Animals treated with the test item did not show statistically significant increases in the incidence of MN-PCE, indicating that graphene did not induce chromosome damage in this in vivo assay. In addition, there were no substantial decreases in the proportion of immature erythrocytes (PCE) indicating that the test item was non-toxic to the bone marrow at the levels tested. For the Comet assay, the lung was collected 3 hours after final dose administration from each animal. A total of 150 cells per animal (149 cells for one animal of group 2) were analyzed for % tail DNA. The positive control, EMS, induced a clear and unequivocal increase in the incidence of % tail DNA. The mean % tail DNA in the negative control group was within the laboratory historical negative control range. The results from both the negative and positive control for the Comet assay confirmed the validity of the assay. Animals treated with graphene did not show any notable increases in the % tail DNA in the lung tissue. Additionally, individual and group mean values for animals treated with graphene were close to the laboratory historical control range. Thus, there was no evidence of an increase in DNA damage attributed to exposure to the test item in this assay.

 

Conclusion

In conclusion, the graphene test material did not induce the formation of micronuclei in polychromatic erythrocytes in the micronucleus test, when administered by nose-only inhalation for 240 minutes for 3 days to male and female rats up to 1.92 mg/L, the maximum respirable practical dose. Also this dose level induced low body weight gain which is indicative of systemic toxicity.

Reference 2

Endpoint:

genetic toxicity in vivo, other

Remarks:

Combined In Vivo Mammalian Erythrocyte Micronucleus Test and Alkaline Comet Assay in Rat

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Feb 2020 to 28 Apr 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes

Remarks:

The study was conducted according to GLP, except for characterization and stability of the test item, which was performed according to established SOPs, controls, and approved test methodologies to ensure integrity and validity of the results generated

Type of assay:

mammalian comet assay

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Canada Inc, St-Constant, QC, Canada.
- Age at study initiation: 49-50 days old
- Weight at study initiation: 181 to 216 (males) and 163 to 184 g (females)
- Assigned to test groups randomly: yes. At the commencement of the study, the weight variation of animals was confirmed to be minimal as it did not exceed ± 20% of the mean weight.
- Housing: Animals were group housed (up to 3 animals of the same dosing group together) in polycarbonate cages containing appropriate bedding equipped with an automatic watering valve. These housing conditions were maintained for the duration of the study. Control group animals were housed on a separate rack from the test item-dosed animals.
- Diet (e.g. ad libitum): ad libitum throughout the study, except during designated procedures
- Water (e.g. ad libitum): Municipal tap water after treatment by reverse osmosis and ultraviolet irradiation was freely available to each animal via an automatic watering system (except during designated procedures).
- Acclimation period: A period of 7 to 8 days was allowed between animal receipt and the start of treatment in order to acclimate the animals to the laboratory environment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C to 25°C
- Humidity (%): 30% to 70%
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

Route of administration:

inhalation

Details on exposure:

TYPE OF INHALATION EXPOSURE: nose only

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Flow-through chamber, chamber type 4 with 2 levels per chamber and 40 breathing ports
- Method of holding animals in test chamber: Restraint tubes
- System of generating particulates/aerosols: Extended Duration Powder Delivery System with Jet Mill and Cycloneb
- Temperature, humidity, pressure in air chamber: 19-25°C, 30-70% relative humidity, and at least 19% O2

TEST ATMOSPHERE
- Brief description of analytical method used: Real-time Qualitative Monitoring: microdust; Aerosol Concentration Analysis, (Substrate/Diameter): Glass fiber/25 mm; Particle Size Analysis (Substrate/Diameter): Stainless steel plates/34 mm

Duration of treatment / exposure:

3 days at 0, 24 and 45 hours (±15 minutes), once daily for up to 240 minutes

Frequency of treatment:

3 days at 0, 24 and 45 hours (±15 minutes), once daily for up to 240 minutes

Dose / conc.:

0 mg/L air

Remarks:

Control

Dose / conc.:

0.5 mg/L air

Dose / conc.:

1 mg/L air

Dose / conc.:

2 mg/L air

Remarks:

according to the acute inhalation study, 2 mg/L air was the maximum respirable practical dose.

No. of animals per sex per dose:

5 per sex per dose

Control animals:

yes

Positive control(s):

Positive control: ethylmethanesulphonate (EMS)
- Justification for choice of positive control(s): EMS is typically administrated to animals used for the comet assay as positive control. The dose level was based on a validated methodology, for which the animals were not expected to show any adverse clinical signs.
- Route of administration: oral gavage
- Doses / concentrations: 200 mg/kg administered once for daily 2 days, with an interval of 21 hours (± 15 minutes).

The first day of dosing was designated as Day 1. If not prepared on the day of dosing, the dose formulations were removed from the refrigerator at least 30 minutes prior to dosing to allow to warm. The doses were given using a syringe with attached gavage tube.

Tissues and cell types examined:

The lungs (groups 1 to 5) were dissected from each main study animal. The collected lung was rinsed with sterile saline 0.9% to remove blood. The left lateral lobe of the lung was cut into two portions, one was used for comet analysis and the other portion was kept for potential histopathology examination.

A total number of 150 cells per animal (149 cells for one animal of Group 2) were analysed for DNA strands breaks.

Details of tissue and slide preparation:

PROCESSING
For all main animals, a portion of the lung was minced in cold homogenizing buffer to create a single cell suspension. The samples were stored on ice until slide preparation and the remaining were discarded.

DETAILS OF SLIDE PREPARATION
Three slides, coated with 1.0 % normal melt agarose, per tissue were prepared from each animal. The cell suspension was mixed with 0.5% low melt agarose (held in a waterbath set to maintain 40ºC). The volume of the cell suspension added to the agarose did not decrease the percentage of low melting agarose by more than 10%. Each sample was dropped on the coated slide. Once solidified, the slides were immersed in complete lysis solution (100 mM EDTA tetrasodium, 2.5 M NaCl and 10 mM Tris hydroxymethyl aminomethane with 1% v/v Triton X-100 and 10% v/v dimethyl sulfoxide added on the day of use), in a light proof box in a refrigerator set to maintain 4°C, until electrophoresis.

The slides were rinsed in purified water to remove excess salts from the slides. They were then transferred to a dry, level platform of a horizontal electrophoresis tank. The slides from each treatment group were positioned systematically to minimize any positional effects. Alkaline buffer (300 mM NaOH and 1 mM EDTA disodium, pH > 13.0) was added to the electrophoresis tank in order to completely cover the slides. The DNA on the slides was allowed to unwind in the alkaline buffer for 20 minutes (± 2 minute). A constant voltage of 16 V (with over current protection at 600 mA) was applied across the tank for 20 minutes (± 1 minute). The temperature of the tank was kept below 10ºC by the use of a recirculating chiller. When electrophoresis was completed, the slides were washed 3 times for 5 minutes with neutralisation buffer (0.4 M Tris hydroxymethyl aminomethane, pH 7.5) and placed in absolute ethanol (previously chilled in a refrigerator set to maintain 4°C) for 30 minutes (± 1 minute). The slides were then allowed to air dry and then stored in humidity controlled lightproof boxes pending slide analysis.

METHOD OF ANALYSIS
After electrophoresis slides were allowed to air dry and then stored in humidity controlled lightproof boxes pending slide analysis. The slides were encoded to minimize potential operator bias. The slides were wet-mounted with 45 μL of propidium iodide (20 μg/mL) and the cells visualized using a fluorescence microscope. Computerized image analysis of selected cells were performed using a Perceptive Instruments COMET IV image analysis system.
Initially, slides were examined visually for any evidence of overt toxicity, e.g. an increase in background debris and/or an increase in the incidence of excessively damaged cells (i.e. ‘hedgehog’ or ‘ghost’ comets). These cells were excluded from analysis, along with cells that had unusual staining artifacts or comets with non-spherical heads. Any observations or comments pertinent to the slide were recorded on a comment sheet.
A total number of 150 cells per animal (149 cells for one animal of Group 2) were analysed for DNA strands breaks.

Evaluation criteria:

CRITERIA FOR BOTH COMET AND MICRONUCLEUS ASSAY

ACCEPTABILITY CRITERIA: concurrent positive and negative controls are within the distribution of the historical control data and positive control induce a statistically significant increase compared with the concurrent negative control; an appropriate number of doses and cells has been analysed
(4000 PCE per animal for micronucleus test or 150 cells per tissue per animal for comet assay); The maximum dose tested is one that allows maximum exposure up to 2000 mg/kg/day for non-toxic compounds, or the limit of solubility, respirability or MTD based on available toxicity data, or the highest dose may also be defined as a dose that produces toxicity in the bone marrow.

CLEARLY POSITIVE
1. At least one of the test item groups exhibits a statistically significant increase in the frequency of MN-PCE or % tail DNA compared with the concurrent negative control.
2. The increase is dose-related when evaluated with an appropriate trend test.
3. Any of the results are outside the distribution of the historical negative control data (e.g. 95% control limits).

Conclusion: The test item is then considered able to induce DNA strand breakage/chromosomal damage or damage to the mitotic apparatus in the tissue studied in this test system.

CLEARLY NEGATIVE
1. None of the test item groups exhibits a statistically significant increase in the frequency of MN-PCE or % tail DNA compared with the concurrent negative control.
2. All results are inside the distribution of the historical negative control data (e.g. 95% control limits).
3. Direct or indirect evidence supportive of exposure of, or toxicity to, the target tissue(s) has been demonstrated.

Conclusion: The test item is then considered unable to induce DNA strand breakage/chromosomal damage or damage to the mitotic apparatus in the tissue studied in this test system.

Statistics:

STATISTICS FOR BOTH COMET AND MICRONUCLEUS ASSAY

All statistical tests were conducted at the 5% significance level. The pairwise comparisons were reported at the 0.1%, 1%, and/or 5% levels. Result was considered to be significant if p ≤ 0.05.

Numerical data collected on scheduled occasions were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation were reported whenever possible.

The numerical data corresponding to the MN-PCE frequency and % tail DNA was statistically evaluated using two separate datasets. The first one included the negative control group and the test item treated groups, whereas, the second one included only the negative control group and the positive control group.

For the first dataset, Levene’s test was used to assess the homogeneity of group variances. If the result of Levene’s test was not found to be significant (p > 0.05), then the groups were compared using an overall one-way ANOVA F-test, otherwise they were compared using Kruskal-Wallis test. If the overall ANOVA F-test or Kruskal-Wallis test was found to be significant (p ≤ 0.05),conducted using Dunnett’s or Dunn’s test, respectively.

For the second dataset, Levene’s test was used to compare the variances of negative and positive control groups. If the result of Levene’s test was not found to be significant (p > 0.05), then the groups were compared using t-test, otherwise, they were compared using Wilcoxon Rank-Sum
test.

Whenever the results of MN-PCE and % tail DNA, corresponding to test item treated groups, reveal significant mean increase when compared to the negative control group, then a dose-related increase trend in means across the negative control group and the test item treated groups would be conducted using Cochran-Armitage’s one-sided.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

There was no mortality, and no remarkable treatment-related clinical signs were noted during the study. Low body weight gain was observed at the highest respirable dose level (actual 1.92 mg/L) which is indicative of systemic toxicity

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Achieved chamber conditions:

Individual mass median aerodynamic diameter (MMAD) of generated aerosols ranged from 2.9 to 5.2 μm. Although this was higher than the targeted 4.0 μm aerosol, generated aerosols were considered acceptable given that the majority of particle size assessments described at least 30% of particles with a diameter < 3.5 μm. On the three consecutive days of inhalation dosing animals in Groups 1 to 4 were exposed to a stable and respirable aerosols of the test item at either 0, 0.547, 1.00 or 1.92 mg/L.

Results of In-life Procedures, Observations, and Measurements

- Mortality: No mortalities occurred during this study.

- Clinical Observations: There were no test item-related clinical signs observed during the dosing and observation period. Black fur staining appeared during the dosing period, and were no longer present at the end of dosing or remained present with slight severity 1 hour post the end of dosing. This observation can be attributed to the nose-only administration of the test item to the animals using a Flow-Through rodent system, and considered non-adverse because of their transient nature.

All other clinical observations were considered unrelated to the test item based on their low and/or sporadic incidence.

- Body Weights and Body Weight Gains: The body weight increased during the pre-treatment period for all the animals. During the treatment period, there was low body weight gain at 1.92 mg/L. The body weight gain values for Days -1 to 3 for the negative control group and test item dose level groups 0.547, 1.0, and 1.92 mg/L were for males: 37.0, 31.8, 35.4 and 23.2 g and for females: 19.8, 19.4, 17.6 and 16.8 g, respectively.

This low body weight gain, observed at the highest test concentration is indicative of systemic toxicity.

Results: % Tail DNA

- All treated animals did not show increases in % tail DNA in the lung tissue relative to control groups

- The group mean values for treated animals as dose levels of 0.547, 1.0 and 1.92 mg/L were 0.92, 0.42, 0.54 % for males and 2.14, 0.89 and 0.49 % for females, respectively. (Values below 95 % negative control limit of 2.40 %).

However, slight but statistically significant increase was obtained with females, group 2 and 3 (2.14 and 0.89 %, respectively), which is not considered biologically relevant for the following reasons: the % tail DNA values of the concurrent negative control are extremely low (0.08% compared to a mean of 1.01% in the historical controls), the results are well below the 95% negative control limit (2.40%) and variability between triplicate slides of 3 out of 5 animals was observed. Thus, the trend test was not considered relevant and therefore not performed.

Thus, the test item did not induce DNA damage, as evaluated by the Comet assay system.

Incidence of Excessively Damaged Cells

Following visual examination of the slides, no evidence of overt toxicity was noted on the slides prepared from the animals treated with the test item (Groups 2, 3 and 4), i.e. there was no substantial increase in background debris and/or increase in the incidence of excessively damaged cells (i.e. non detectable cell nuclei (NDCN), hedgehog or ghost comets) compared to the negative control Group 1. The group mean incidence for the negative control was 5.8 for males and 0.7 for females and for the test item treated groups between 8.3 to 8.7 for males and 9.0 to 11.9. The low value obtained with the females negative control might be due to normal variation as all the values remain below the positive controls and there is no increase in % tail DNA.

Conclusions:

In conclusion, the graphene test material did not show evidence of induction of DNA damage in the lung in the in vivo comet assay, when administered by nose-only inhalation for 240 minutes for 3 days to male and female rats up to 1.92 mg/L, the maximum respirable practical dose. Also this dose level induced low body weight gain which is indicative of systemic toxicity.

Executive summary:

Study design

The objective of this GLP-study was to determine the potential genotoxicity of graphene when given by nose-only inhalation for 240 minutes for 3 days in rats using the bone marrow micronucleus test and the comet assay with the lung using OECD 474 and 489 guidelines.

In the study, rats were dosed using target concentrations of 0.5, 1 and 2 mg/L of graphene (5 animals/sex/group). The dose levels were selected based on the results of acute inhalation toxicity study (Zabaiou, 2020), where it was determined that the test item at 2 mg/L is the maximum respirable practical dose. 20 mg/mL (10 mL/kg) Ethyl methanesulfonate (EMS) or cyclophosphamide was used as positive controls (3 male animals). Bone marrow and lung were collected 3 hours after the last treatment.

 

Results

The generated aerosols of graphene were considered to be within the respirable range for the rats and the target concentrations were achieved during the three consecutive days of dosing by inhalation. There was no mortality, and no remarkable treatment-related clinical signs were noted during the study. Low body weight gain was observed at the highest respirable dose level (actual 1.92 mg/L) which is indicative of systemic toxicity. A total of 4000 immature polychromatic erythrocytes (PCEs) were evaluated for presence of micronuclei indicative of chromosome damage. In addition, the proportion of immature erythrocytes in the total of at least 500 erythrocytes per animal was assessed as a measure of potential bone marrow toxicity. The proportion of polychromatic erythrocytes and the frequencies of micronucleated polychromatic erythrocytes (MN-PCE) in the negative control group were within the laboratory historical negative control range. The positive control, cyclophosphamide, induced a clear and unequivocal increase in micronuclei. The results from both the negative and positive controls confirmed the validity of the assay. Animals treated with the test item did not show statistically significant increases in the incidence of MN-PCE, indicating that graphene did not induce chromosome damage in this in vivo assay. In addition, there were no substantial decreases in the proportion of immature erythrocytes (PCE) indicating that the test item was non-toxic to the bone marrow at the levels tested. For the Comet assay, the lung was collected 3 hours after final dose administration from each animal. A total of 150 cells per animal (149 cells for one animal of group 2) were analyzed for % tail DNA. The positive control, EMS, induced a clear and unequivocal increase in the incidence of % tail DNA. The mean % tail DNA in the negative control group was within the laboratory historical negative control range. The results from both the negative and positive control for the Comet assay confirmed the validity of the assay. Animals treated with graphene did not show any notable increases in the % tail DNA in the lung tissue. Additionally, individual and group mean values for animals treated with graphene were close to the laboratory historical control range. Thus, there was no evidence of an increase in DNA damage attributed to exposure to the test item in this assay.

 

Conclusion

In conclusion, the graphene test material did not show evidence of induction of DNA damage in the lung in the in vivo comet assay, when administered by nose-only inhalation for 240 minutes for 3 days to male and female rats up to 1.92 mg/L, the maximum respirable practical dose. Also this dose level induced low body weight gain which is indicative of systemic toxicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro - CA

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 (CMC) 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.

As mentioned in the OECD TG 473, specific adaptations testing manufactured nanoparticles are recommended, but not specified. To adress this recommendation in line with the Guideline, additional investigations were performed examining cellular uptake of graphene.

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.

 

Genetic toxicity in vitro - HPRT

Study design

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

Results

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.

Conclusion

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.

 

Genetic toxicity in vivo Comet Assay and Micronuleus Assay

Study design

The objective of this GLP-study was to determine the potential genotoxicity of graphene when given by nose-only inhalation for 240 minutes for 3 days to rats using the bone marrow micronucleus test and the comet assay with the lung using OECD TG 474 and 489. The dose levels were selected based on the results of acute inhalation toxicity study (Zabaiou, 2020), where it was determined that the test item at 2 mg/L is the maximum respirable practical dose. The animals were dosed by nose-only inhalation for 240 minutes for 3 days. Bone marrow and lung were collected 3 hours after last treatment.

Results

The generated aerosols of graphene were considered to be within the respirable range for the rats and the target concentrations were achieved during the three consecutive days of dosing by inhalation. There was no mortality, and no remarkable treatment-related clinical signs were noted during the study. Low body weight gain was observed at the highest respirable dose level (actual 1.92 mg/L) which is indicative of systemic toxicity. A total of 4000 immature polychromatic erythrocytes (PCEs) were evaluated for presence of micronuclei indicative of chromosome damage. In addition, the proportion of immature erythrocytes in the total of at least 500 erythrocytes per animal was assessed as a measure of potential bone marrow toxicity. The proportion of polychromatic erythrocytes and the frequencies of micronucleated polychromatic erythrocytes (MN-PCE) in the negative control group were within the laboratory historical negative control range. The positive control, cyclophosphamide, induced a clear and unequivocal increase in micronuclei. The results from both the negative and positive controls confirmed the validity of the assay. Animals treated with the test item did not show statistically significant increases in the incidence of MN-PCE, indicating that graphene did not induce chromosome damage in this in vivo assay. In addition, there were no substantial decreases in the proportion of immature erythrocytes (PCE) indicating that the test item was non-toxic to the bone marrow at the levels tested. For the Comet assay, the lung was collected 3 hours after final dose administration from each animal. A total of 150 cells per animal (149 cells for one animal of group 2) were analyzed for % tail DNA. The positive control, EMS, induced a clear and unequivocal increase in the incidence of % tail DNA. The mean % tail DNA in the negative control group was within the laboratory historical negative control range. The results from both the negative and positive control for the Comet assay confirmed the validity of the assay. Animals treated with graphene did not show any notable increases in the % tail DNA in the lung tissue. Additionally, individual and group mean values for animals treated with graphene were close to the laboratory historical control range. Thus, there was no evidence of an increase in DNA damage attributed to exposure to the test item in this assay.

Conclusion

In conclusion, the test item did not induce the formation of micronuclei in polychromatic erythrocytes in the micronucleus test or show evidence of induction of DNA damage in the lung in the in vivo comet assay, when administered by nose-only inhalation for 240 minutes for 3 days to male and female rats up to 1.92 mg/L, the maximum respirable practical dose. Also this dose level induced low body weight gain which is indicative of systemic toxicity.

Justification for classification or non-classification

Available genetic toxicity in vitro studies OECD TG 473 and 476 indicate no potential for genotoxicity. The key study combining bone marrow micronucleus test and comet assy (OECD 474 and 489) indicate also no potential for genotoxicity, however, the highest dose level (1.92 mg/L) induced low body weight gain which is indicative of systemic toxicity.

Based on these results no classification for genotoxicity is triggered in accordance with Regulation (EC) No 1272/2008.