Niobium doped titanium dioxide

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:

September 17, 2018 - September 27, 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)

Version / remarks:

21st July 1997

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:

Niobium doped titanium dioxide

EC Number:

951-670-4

Molecular formula:

Nb:TiO2

IUPAC Name:

Niobium doped titanium dioxide

Test material form:

solid

Method

Target gene:

HIS operon (S. thyphimurium)
TRP operon (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:

Type and composition of metabolic activation system:
- source of S9 :
Post-mitochondrial S9 fraction derived from rat liver homogenate
- method of preparation of S9 mix:
Male Wistar rats, Crl:WI (HAN) (Charles River, Germany), aged 6-8 weeks were pretreated with 3-Naphthoflavone (100 mg/kg body weight) and Phénobarbital (80 mg/kg body weight) diluted in Miglyol 812 and administered orally once daily on three consecutive days. About 16 hours before sacrifice, the rats remained without food. One day after the last administration of P-Naphthoflavone/Phenobarbital the animals were killed, their livers removed and homogenized in ice-cold 0.15 M KC1 (3 mL KC1 per g liver wet-weight). The homogenate was spun for 10 minutes at 9000 rpm (8784 x g, Biofuge 28 RS) and +4°C. The supernatant fluid (S9) was decanted, transferred to sterile tubes and stored in liquid nitrogen.
- concentration or volume of S9 mix and S9 in the final culture medium:
The S9 mix used contained 10% S9 in the 1st and 20% S9 in the 2nd series, respectively.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability):
Each S9 batch was tested for its metabolic activity using specific substrates, requiring different enzymes of the P450-isoenzyme family. The mutagenicity of 2-aminoanthracene, benzo[a]pyrene, and 3-methylcholanthrene is thus determined once for each S9 batch.

Test concentrations with justification for top dose:

Due to limited solubility, the test item was applied as a homogenous suspension at concentrations ≥ 15.8 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 0.25% aqueous methylcellulose

- Justification for choice of solvent/vehicle: The test item was not soluble in any standard solvent used in our laboratory so far. However, a homogenous suspension was achieved with this specific batch using 0.25% aqueous hydroxypropyl methylcellulose (Methocel® K4M) as vehicle at a maximum concentration of 100 µL/plate.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Daunomycin

Remarks:

without S9 mix, TA98

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix, E.coli

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 mix, TA100, TA1535

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix, TA1537

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with S9 mix, TA98, TA100, TA1535, TA 1537, E.coli

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 3
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: The incubation of plates was performed at 36 - 38 °C for 2 days.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

Evaluation criteria:

A test material was to be defined as positive or mutagenic in this assay if
• the assay is considered valid and
• a biologically relevant increase in the mean number of revertants above a threshold of 2-fold (TA 98, TA 100, WP2 uvrA) or 3-fold (TA 1535, TA 1537) as compared to the concurrent negative controls is observed
• an increase exceeding the threshold at only one concentration is considered as biologically meaningful if reproduced in a second independent experiment
• a concentration-dependent increase is considered biologically meaningful if the threshold is exceeded at more than one concentration
A test material is defined as negative or non-mutagenic in this assay if
• the assay is considered valid and
• none of the above-mentioned criteria are met
Whenever colony counts remain within the historical range of negative controls, such increases are considered as biologically not meaningful. In general, two series of experiments must be performed. However, there is no requirement for verification of a clear positive response

Statistics:

none

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

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

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

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

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

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

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

True negative controls validity:

not examined

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

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Due to limited solubility, the test item was applied as a homogenous suspension at concentrations ≥15.8 µg/plate.
- Precipitation and time of the determination: yes

STUDY RESULTS
Ames test:
- Signs of toxicity : no
- Individual plate counts : see attachment
- Mean number of revertant colonies per plate and standard deviation : see attachment

HISTORICAL CONTROL DATA
- see attachment

Applicant's summary and conclusion

Conclusions:

The test item was not mutagenic under the described experimental conditions.

Executive summary:

A study according OECD TG 471 was performed to examine the mutagenic activity of the test item in an in vitro bacterial reverse mutation test employing Salmonella typhimurium TA 98, TA 100. TA 1535 and TA 1537 and Escherichia coli WP2 uvrA as indicator organisms.

The plate incorporation test with and without addition of liver S9 mix from Phenobarbital/beta Naphthoflavone-pretreated rats was used. In this study, two experimental series were performed. In the experiments with S9 mix, 10% and 20% S9 in the S9 mix were used. Treatments of all tester strains were performed using formulations prepared in 0.25% aqueous hydoxypropyl methylcellulose (Methocel® K4M) in the absence and in the presence of S9 mix using final concentrations between 5 and 5000 µg/plate plus untreated, vehicle and positive controls. Due to limited solubility the test item was applied as a homogenous suspension at concentrations ≥15.8 µg/plate. The mean numbers of revertant colonies of the current negative controls were within the range of historical negative control values and were not significantly different from the untreated controls. The strain-specific positive controls, namely daunomycin, sodium azide, 4-nitroquinolin-N-oxide and 9-aminoacridine in the absence of S9 mix yielded the expected mutant frequencies that were greatly exceeding the negative controls. The genotype of the tester strains used was thus confirmed. 2-Aminoanthracene which requires metabolic activation was strongly mutagenic. This indicates that the exogenous metabolizing system used in the present investigation (S9 mix) was active. Thus, the requirements predetermined in the study plan of this report have been met in total and the study is considered valid. Following treatment with the substance, the test material was still visible as a homogenously distributed suspension on the agar plates at the end of exposure at concentrations ≥ 1580 µg/plate. No toxicity to the bacteria was observed. There were no relevant increases in revertant numbers observed after exposure in the absence and presence of S9 mix. According to the criteria for negative and positive results predetermined the test item was not mutagenic under the described experimental conditions.