branched and cross-linked multi-walled carbon nanotubes

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 08 March 2021
Experimental completion date: 08 July 2021

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 using the Hprt and xprt genes)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Specific details on test material used for the study:

Identification: Athlos™ 200 Carbon Nano Structures
EC Number: 951-407-3
Batch Number: V20169
Purity: > 97%
Molecular Weight: 12
Expiry Date: 17 June 2025
Appearance: Black pellets
Storage Conditions: Room temperature in the dark
A correction for the impurity of the test item (3%) was applied to the test item formulations

Target gene:

Hprt and xprt

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Cell Line
The V79 cell line has been used successfully in in vitro experiments for many years. 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%) make it an appropriate cell line to use for this study type. The cells have a stable karyotype with a modal chromosome number of 22 (Howard-Flanders, 1981).
The V79 cell stocks were obtained from Harlan CCR in 2010 and originated from Labor für Mutagenitätsprüfungen (LMP); Technical University; 64287 Darmstadt, Germany.

Cell Culture
The stock of cells is stored in liquid nitrogen. For use, a sample of cells will be removed before the start of the study and grown in Eagles Minimal Essential (MEM) (supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10% fetal bovine serum (FBS)) at approximately 37 C with 5% CO2 in humidified air. Master stocks of cells were tested and found to be free of mycoplasma.

Cell Cleansing
Cell stocks spontaneously mutate at a low but significant rate. Before a stock of cells is frozen for storage the number of pre-existing HPRT-deficient mutants must be reduced. The cells are cleansed of mutants by culturing in HAT medium for four days. 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). After four days in medium containing HAT, the cells are passaged into HAT free medium and grown for four to seven days. Bulk frozen stocks of these “HAT” cleansed cells are frozen down prior to use in the mutation studies, with fresh cultures being removed from frozen before each experiment.

Metabolic activation:

with and without

Metabolic activation system:

The S9 Microsomal Enzyme Fraction was purchased from Moltox and Lot no 4370 with the expiry date of 24 November 2022 was used in this study. The S9 was pre-tested for acceptability by the supplier prior to purchase and was supplied with a relevant “Quality Control & Production Certificate”. The protein content was adjusted to 20 mg/ml prior to use.
The S9 mix was prepared by mixing S9 with a phosphate buffer containing NADP (5 mM), G6-P (5 mM), KCl (33 mM) and MgCl2 (8 mM) to give a 20% or 10% S9 concentration. The final concentration of S9 when dosed at a 10% volume of S9-mix was 2% for the Preliminary Toxicity Test and the Main Experiment.

Test concentrations with justification for top dose:

0, 0.02, 0.05, 0.09, 0.19, 0.375, 0.75, 1.5, 3, 6 μg/mL

The molecular weight of the test item was 12, therefore the maximum recommended concentration 120 μg/mL which was equivalent to 10 mM. The purity of the test item was 97% and was therefore accounted for when formulating the dosing solutions. The test item was insoluble in culture medium at 12 mg/mL and acetone at 6 mg/mL but could be formulated in DMSO at 0.6 μg/mL to provide a suspension suitable for dosing. Therefore, the maximum practical dose level for testing was 6 μg/mL

Precipitate was observed at and above 0.19 μg/mL when the formulations were dose into culture media. Based on this observation, the maximum concentration selected for the preliminary toxicity test was limited to 0.75μg/mL.

The concentrations used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by test item precipitate as recommended by the OECD 476 guideline.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: Dimethyl benzanthracene (DMBA)

Remarks:

With metabolic activation

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

Preliminary Cytotoxicity Test
Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient cells for use in the test. The preliminary cytotoxicity test was performed on cell cultures plated out at 2 x 10^6 cells/225 cm2 flask 2 days before dosing. This was demonstrated to provide at least 20 x 10^6 available for dosing in each flask using a parallel flask, counted at the time of dosing. On dosing, the growth media was removed and replaced with serum-free Minimal Essential Medium (MEM). One flask per concentration was treated for 4-hours without metabolic activation and for 4-hours with metabolic activation (2% S9). The concentrations of test item used were 0, 0.003, 0.006, 0.01, 0.02, 0.05, 0.09, 0.19, 0.38 and 0.75 μg/mL. The maximum dose level selected was limited to 0.75 μg/mL based on the precipitate observations made in the solubility test.
Exposure was for 4 hours at approximately 37 °C with a humidified atmosphere of 5% CO2 in air, after which the cultures were washed twice with phosphate buffered saline (PBS) before being detached from the flasks using trypsin. Cells from each flask were suspended in MEM with 10% FBS, a sample was removed from each concentration group and counted using a Coulter counter. For each culture, 200 cells were plated out into three 25 cm2 flasks with 5 mL of MEM with 10% FBS and incubated for 6 days at approximately 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air. The cells were then fixed and stained and total numbers of colonies in each flask counted to give relative survival (RS). A comparison of the test item to vehicle control relative survivals gave the relative toxicity of each test item concentration.
Results from the preliminary cytotoxicity test were used to select the test item concentrations for the mutagenicity experiment.

Mutagenicity Test
Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient cells for use in the test. Cells were seeded at 2 x 10^6 cells/225 cm2 flask two days being exposed to the test or control items. This was demonstrated to provide at least 20 x 106 available for dosing in each flask using a parallel flask. Duplicate cultures were set up, both in the presence and absence of metabolic activation, with eight test item concentrations in the absence of S9 and six in the presence of S9, and vehicle and positive controls. Treatment was for 4 hours in serum free media (MEM) at approximately 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air.
At the end of the treatment period the flasks were washed twice with PBS, detached from the flasks with trypsin and the cells suspended in MEM with 10% FBS. A sample of each concentration group cell suspension was counted using a Coulter counter. Cultures were plated out at 2 x 10^6 cells/flask in a 225 cm2 flask to allow growth and expression of induced mutants, and in triplicate in 25 cm2 flasks at 200 cells/flask to obtain the cloning efficiency,
for an estimate of cytotoxicity at the end of the exposure period. Cells were grown in MEM with 10% FBS and incubated at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air.
Cytotoxicity flasks were incubated for 6 days then fixed with methanol and stained with Giemsa. Colonies were manually counted to give relative survival (RS). A comparison of the test item to vehicle control relative survivals gave the relative toxicity of each test item concentration.
During the 7 Day expression period the cultures were sub-cultured and maintained on days 2
and 5 to maintain logarithmic growth. At the end of the expression period the cell monolayers were detached using trypsin, cell suspensions counted using a Coulter counter and plated out as follows:
i) In triplicate at 200 cells/25 cm2 flask in 5 mL of MEM with 10% FBS to determine cloning efficiency. Flasks were incubated for 6 days, fixed with methanol and stained with Giemsa. Colonies were manually counted; counts were recorded for each culture and the percentage cloning efficiency for each dose group calculated.
ii) At 2 x 10^5 cells/petri dish (ten replicates per group) in MEM with 10% FBS supplemented with 11 μg/mL 6-Thioguanine (6-TG), to determine mutant frequency. The dishes were incubated for 7 days at 37 °C in an incubator with humidified atmosphere of 5% CO2 in air, then fixed with methanol and stained with Giemsa. Mutant colonies were manually counted and recorded for each dish.
The percentage cloning efficiency and mutant frequency per survivor were calculated for each dose group.
Fixation and staining of all flasks/petri dishes was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes.

Evaluation criteria:

Please see "any other information on materials and methods"

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

Positive controls validity:

valid

Additional information on results:

Preliminary Cytotoxicity Test
The test item concentrations used were 0, 0.003, 0.006, 0.01, 0.02, 0.05, 0.09, 0.19, 0.38 and 0.75 μg/mL. The maximum concentration tested was the limited by test item precipitate based on the observations made in the preliminary toxicity test. This follows the recommendations in the OECD 476 guideline which states the lowest precipitating dose level should be maximum concentration tested.
A precipitate of the test item was observed at the end of exposure at and above 0.19 μg/mL in both the 4-hour exposure groups.
From the results of the individual flask counts it can be seen that there was consistent dose-related reduction in the relative survival (RS) in the absence of metabolic activation (S9). In the presence of S9 there was some toxicity observed at 0.09 and 0.19 μg/mL around the lowest precipitating dose level.

Mutagenicity Test – Main Experiment
At the end of the exposure period, precipitate of the test item was observed at and above 0.20 μg/mL in the absence of metabolic activation and at and above 0.10 μg/mL in the presence of metabolic activation. The dose levels of 0.00313, 0.00625 and 0.40 μg/mL in the absence of S9 were discarded at this point as they were surplus to requirements. The dose levels of 0.2 and 0.4 μg/mL in the presence of metabolic activation were discarded prior to Day 7 plating since only one precipitating dose level was required.
In the Day 0 relative survival and Day 7 cloning efficiencies for the exposure groups in the absence and presence of metabolic activation there was no marked concentration-related reductions in the relative survival values in either the absence or presence of metabolic activation. There was also no evidence of any reductions in the Day 7 cloning efficiencies in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred.
The test item did not induce any statistically significant increases in mutant frequency at any of the concentrations in the absence of metabolic activation. There were also no statistically significant concentration related increases when evaluated with a trend test, and all of the values observed were within the historical control range and 95% control limits for solvent controls.
In the presence of metabolic activation there was a small but statistically significant increase in mutant frequency at 0.1 μg/mL. The mutant frequency at 0.1μg/mL was within the historical control range and the 95% control limits for solvent controls. It was at a lower level than mutant frequencies seen in the 4-hour exposure group in the presence of S9 where there were no statistically significant increases but because the mutant frequency at 0.1 μg/mL in the absence of S9 was being compared to a relatively low vehicle control value it was statistically significant. There was no statistically significant concentration related increase when evaluated with a trend test. The response was therefore considered to be of no toxicological relevance.
The vehicle control values were all considered to be within an acceptable range, and the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected.

Conclusions:

The test item did not induce any toxicologically significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation. Athlos™ 200 Carbon Nano Structures was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.

Executive summary:

Introduction
The purpose of this study is to assess the potential mutagenicity of a test item, supplied by the Sponsor, on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line.

Methods
Chinese hamster (V79) cells were treated with the test item at up to six concentrations, in duplicate, together with solvent (dimethyl sulfoxide (DMSO)) and positive controls in the absence and presence of metabolic activation (S9).
The concentrations used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be the limited by test item precipitate, as recommended by the OECD 476 guideline. The concentrations of test item plated for relative survival, cloning efficiency, and expression of mutant colonies were as follows:

4-hour without S9: 0, 0.0125, 0.025, 0.05, 0.1, 0.2 (Final concentration of test item (μg/mL))
4-hour with S9 (2%):0, 0.0125, 0.025, 0.05, 0.1 (Final concentration of test item (μg/mL))

Results
The solvent (DMSO) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus.
The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolising system.
The test item did not induce any statistically significant increases in mutant frequency at any of the concentrations in the absence of metabolic activation. There were also no statistically significant concentration related increases when evaluated with a trend test, and all of the values observed were within the historical control range and 95% control limits for solvent controls.
In the presence of metabolic activation there was a small but statistically significant increase in mutant frequency at 0.1 μg/mL. The mutant frequency at 0.1μg/mL was within the historical control range and 95% control limits for solvent controls and was set against a low solvent control value. There was no statistically significant concentration related increase when evaluated with a trend test. The response was therefore considered to be of no toxicological relevance.

Conclusion
The test item did not induce any toxicologically significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation. Athlos™ 200 Carbon Nano Structures was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)