Single Wall Carbon Nanotubes (SWCNT)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 June 2016 - 14 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guidelines for Testing of Chemicals No. 471 (1997) "Bacterial Reverse
Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tin dioxide

EC Number:

242-159-0

EC Name:

Tin dioxide

Cas Number:

18282-10-5

Molecular formula:

O2Sn

IUPAC Name:

Tin Dioxide

Test material form:

solid: nanoform

Details on test material:

Sample Reference: W0167

Method

Target gene:

Histidine or Tryptophan locus.

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

10% liver S9 in standard co-factors

Test concentrations with justification for top dose:

The maximum concentration was 5000 μg/plate (the maximum recommended dose level). Eight concentrations of the test item
(1.5, 5, 15, 50, 150, 500, 1500 and 5000 g/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Vehicle / solvent:

Distilled water.
In solubility checks performed in–house, the test item was noted to be insoluble in sterile
distilled water at 12.5, 25 and 50 mg/mL, dimethyl sulphoxide at 25 and 50 mg/mL and
dimethyl formamide and polyethylene glycol 400 at 50 mg/mL. The test item formed the
best doseable suspension in sterile distilled water at a maximum concentration of
12.5 mg/mL, therefore, this solvent was selected as the vehicle.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

0.4 mL of the appropriate concentration of test item or solvent vehicle or 0.1 mL of
appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented
media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate
buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative
(untreated) controls were also performed on the same day as the mutation test. Each
concentration of the test item, appropriate positive, vehicle and negative controls, and each
bacterial strain, was assayed using triplicate plates. For metabolic activation, 0.5 mL of S9-mix was added to the
molten, trace amino-acid supplemented media instead of phosphate buffer.
All of the plates were incubated at 37 ± 3 C for approximately 48 hours and scored for the
presence of revertant colonies using an automated colony counting system. The plates were
viewed microscopically for evidence of thinning (toxicity). Manual counts were performed
at and above 1500 μg/plate because of test item precipitation. Several further manual counts
were also performed due to bubble interference and artefacts on the plates, thus distorting the
actual plate count.

Evaluation criteria:

A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
A reproducible increase at one or more concentrations.
Biological relevance against in-house historical control ranges.
Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996).

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). A black test item precipitate was noted by eye at and above 5 g/plate, this observation did not prevent the scoring of revertant colonies.

There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). Small, statistically significant increases in TA1535 revertant colony frequency were observed in the first mutation test at 50, 150 and 1500 μg/plate in the absence of S9-mix only. These increases were considered to be of no biological relevance because there was no clear evidence of a dose-response relationship or reproducibility and the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for the tester strain. The response was more likely due to a slightly low vehicle control count.

Applicant's summary and conclusion

Conclusions:

The test item was considered to be non-mutagenic under the conditions of this test.

Executive summary:

A bacterial reverse mutation test was performed according to OECD TG 471.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with suspensions of the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 g/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 μg/plate. Six test item concentrations were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology.

There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in both mutation tests.

There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix), in both experiments.

Small, statistically significant increases in TA1535 revertant colony frequency were observed in the first mutation test at 50, 150 and 1500 μg/plate in the absence of S9-mix only. These increases were considered to be of no biological relevance because there was no clear evidence of a dose-response relationship or reproducibility and the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for the tester strain. The response was more likely due to a slightly low vehicle control count.

The vehicle (sterile distilled water) control plates gave counts of revertant colonies generally within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Test item was considered to be non-mutagenic under the conditions of this test.