Titanium nitride

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:

Experimental phase: 24 May 2018 to 14 June 2018. Report issue: 21 August 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Two deviations were noted which were not considered to have had any impact on the result or integrity of the study.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Titanium nitride

EC Number:

247-117-5

EC Name:

Titanium nitride

Cas Number:

25583-20-4

Molecular formula:

NTi

IUPAC Name:

titanium nitride

Test material form:

solid: nanoform

Method

Target gene:

Histidine for Salmonella
Trptophan for E. coli

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:

Rat liver microsomal enzymes (S9) from rats administered Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1: Strains TA98, TA100, TA 1535, TA1537 and WP2uvrA
0, 52, 164, 512, 1000 and 2500 µg/plate

Experiment 2: Strains TA98, TA100, TA 1535, TA1537 and WP2uvrA
0, 52, 164, 512, 1000 and 2500 µg/plate

No correction was made for the purity/composition of the test item.

The test concentrations for experiments 1 and 2 were selected based on the results of a dose range-finder test where the test substance was initially tested in the tester strains TA100 and WP2uvrA at concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate in the absence and presence of S9-mix. The test item precipitated at dose levels of 1600 µg/plate and above. No effect on background lawn was observed and there was no biologically relevant decrease in in the number of revertants.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water (Milli-Q water, Millipore Corp., Bedford, MA., USA).

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA98

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA100

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without metabolic activation

Controls 3

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation

Controls 4

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Acridine Mutagen ICR-191

Remarks:

Without metabolic activation

Controls 5

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without metabolic activation

Controls 6

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without metabolic activation

Controls 7

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA98

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation

Controls 8

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA100

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation

Controls 9

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation

Controls 10

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation

Controls 11

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation) for experiment 1 and preincubation for experiment 2.

DURATION
- Preincubation period: 30 ± 2 minutes (second preincubation experiment only)
- Exposure duration: 48 ± 4 hours

NUMBER OF REPLICATIONS: Triplicate for treatment.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:

a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control.

b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:

a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control.

b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed in in the absence of S9 at 2500 µg/plate (highest concentration tested) in the pre-inucubation assay (the second experiment).

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

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

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

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

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed in in the absence of S9 at 2500 µg/plate (highest concentration tested) in the pre-inucubation assay (the second experiment).

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

First Experiment:  Direct Plate Assay

Precipitate

Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 5000 µg/plate and at 1600 µg/plate and above at the end of the incubation period.

Toxicity

No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

Second Experiment:  Pre-Incubation Assay

Precipitate

Precipitation of the test item on the plates was not observed at the start of the incubation period. At the end of the incubation period precipitate was observed at the top dose level of 2500 µg/plate.

Toxicity

Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strains TA1537 and TA98 in the absence of S9-mix at the highest tested concentration.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that Titanium Nitride (Nanoform) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Introduction

The study was to determine the potential of Titanium Nitride (Nanoform) to induce reverse mutations in several strains of Salmonellatyphimurium and in Escherichia coli (E. coli) strain WP₂uvrA. The method followed was designed to be compatible with OECD Guideline 471. Genetic Toxicology: Bacterial Reverse Mutation Test. (Adopted July 21, 1997). 

 

Method

The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP₂uvrA in the direct plate assay. The test item precipitated on the plates at dose levels of 1600 μg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose-range finding test were reported as part of the first mutation assay.

In the first mutation experiment, the test item was tested up to concentrations of 2500 µg/plate in the strains TA1535, TA1537 and TA98. The test item precipitated on the plates at the highest dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the second mutation experiment, the test item was tested up to concentrations of 2500 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP₂uvrA in the pre-incubation assay. The test item precipitated on the plates at the top dose of 2500 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strains TA1537 and TA98 in the absence of S9-mix at the highest tested concentration.   

Result

The test item did not induce a significant dose-related increase in the number of revertant (His⁺) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant colonies in tester strain WP₂uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

Results from the positive and negative controls were within acceptable ranges thus confirming that the assay was responding as expected. 

Conclusion

Based on the results of this study it is concluded that Titanium Nitride (Nanoform) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.