Niobium oxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2003-01-02 to 2003-03-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Niobium oxide

EC Number:

234-808-1

EC Name:

Niobium oxide

Cas Number:

12034-57-0

Molecular formula:

NbO

IUPAC Name:

niobium oxide

Test material form:

solid

Details on test material:

- Lot/batch No. of test material: SNB10OG200277M
- Expiration date of the lot/batch: 2003-12-31
- Purity: >99%
- Description: black, odourless powder
- Storage condition of test material: dry, room temperatue, closed container

Specific details on test material used for the study:

- Treatment of test material prior to testing: The test item was suspended in dimethylsulfoxide prior to use after crushing with a pestle and mortar to a fine dust, because there is no other suitable solvent. The suspension was carefully mixed immediately before administration. The homogeneity was proved visually.

Method

Target gene:

Salmonella typhimurium TA1535, TA1537, TA 100, TA 98: Histidine locus
Escherichia coli WP2uvrA: Tryptophan locus

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Prof. Bruce N. Ames (University of California, Berkely, USA)

MEDIA USED
- Nutrient medium: 15 g Nutrient broth (SIFIN GmbH, D-13088 Berlin) per litre
- Selective Agar: minimal glucose agar plates
- Overlay Agar: 9 g agar agar (Merck, D-64293 Darmstadt), 5 g NaCl, 100 mL sterile 0.5 mM Histidine/Biotine (Merck, D-64293 Darmstadt) solution per litre

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, 38124 Braunschweig, Germany)

MEDIA USED
- Nutrient medium: 15 g Nutrient broth (SIFIN GmbH, 13088 Berlin, Germany) per litre
- Selective Agar: minimal glucose agar plates
- Overlay Agar: 9 g agar agar (Merck, D-64293 Darmstadt), 5 g NaCl, 10 mL sterile Tryptophan solution (1 mg/ml; Merck, D-64293 Darmstadt) per litre.

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

TA 100 and the E. coli strain were tested in dose range finding study. A direct plate incorporation test was carried out, using concentrations of the test item of 5.0, 1.0, 0.5, 0.1, 0.05 and 0.01 mg/plate.
Based on the results the follwing five concentrations were chosen for the main experiment: 5.0, 1.0, 0.5, 0.1 and 0.05 mg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 100 and TA 1535 without S9, 5 µg/plate

Controls 2

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 without S9, 100 µg/plate

Controls 3

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA 98 without S9, 5 µg/plate

Controls 4

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

E.coli WP2uvrA, without S9, 1.3 µg/plate

Controls 5

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

All strains, with S9, S. tymphimurium: 5.0 µg/plate, E.coli: 50.0 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation, Experiment I); pre-incubation (in suspension, Experiment II)

EXPERIMENTAL PERFORMANCE
- Experiment I:
The following were added to sterile tubes containing 2 ml of top agar: 0.1 ml of test item solution (or positive control or solvent) solution, 0.1 ml of bacteria culture, 0.5 ml of S9 mix (or 0.2 M phosphate buffer). The contents of each tube were mixed and added to a Petri dish containing minimal agar. Once the top agar had set, the dishes were inverted and incubated at 37 °C for 48 h.
- Experiment II:
The following were added to sterile tubes: 0.1 ml of test item solution (or positive control or solvent) solution, 0.1 ml of bacteria culture, 0.5 ml of S9 mix (or 0.2 M phosphate buffer). Each tube was fitted with a sterile cap and the contents were mixed followed by incubation at 37 °C for 20 min. Two ml of top agar were added to each tube and following mixing the contents were added to petri dishes containing minimal agar. When the top agar had set, the dishes were inverted and incubated at 37 °C for 48 h.

SCORING
Revertant colonies were scored manually at the end of the incubation period.

DURATION
- Pre-incubation period:(Experiment II): 20 min at 37 °C
- Exposure duration: 48 h at 37°C

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Plate incorporation test with S. typhimurium TA 100 and E. coli WP2 uvrA
- Determination of spontaneous revertants and condition of the bacterial background lawn

Evaluation criteria:

- Positive Result:
A Test is considered to be positive if the test item induces dose related increases in numbers of revertants scored in two separate experiments and these
increases are deemed to be of biological relevance. Reproducible increases at one experimental point may also be indicative of a positive response. For a biologically relevant response the number of revertants is expected to be at least the double of spontaneous reversion.
- Negative Result:
A test item producing neither a dose related and reproducible increase in the number of revertants nor a reproducible positive response at any experiment point is considered to be non-mutagenic in this test system.
- Results of Positive Controls:
The positive controls should markedly increase the number of revertant colonies in all bacterial strains with and without metabolic activation to demonstrate the effective performance of each assay.

Statistics:

The results are presented as individual plate counts. In addition mean values and standard deviations are calculated for each strain and experimental point. As unequivocal results were obtained in both experiments a statistical evaluation was not deemed to be necessary.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA 1535 and 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 2

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

Additional information on results:

For detailed results please refer to Table 1 in box "Any other information on results incl. tables".

Any other information on results incl. tables

Table 1: Summarized Results

Bacteria tester strain	Metabolic activation	Exp.	Mean numbers of revertants								Evaluation
			Controls			mg test item per plate
			UTC	VC	PC	5.0	1.0	0.5	0.1	0.05
Salmonella typhimurium
TA1535	no	1^st	19.0	21.0	1269.3	18.3	19.0	19.0	20.7	19.0	-
	no	2^nd	18.0	18.3	1792.0	12.0	17.3	18.7	16.0	16.7	-
	yes	1^st	25.0	27.0	282.7	22.3	22.0	22.7	21.3	24.3	-
	yes	2^nd	18.7	21.0	309.3	21.0	19.0	21.7	19.7	19.7	-
TA1537	no	1^st	8.7	11.7	2560.0	8.3	7.7	7.3	7.0	9.3	-
	no	2^nd	14.7	14.0	1600.0	13.3	13.0	12.7	11.0	10.7	-
	yes	1^st	12.7	13.0	389.3	12.7	11.3	11.0	12.0	13.7	-
	yes	2^nd	14.3	12.7	389.3	11.3	12.7	11.3	12.0	9.3	-
TA 98	no	1^st	24.3	20.0	720.0	18.3	15.3	21.3	17.7	18.3	-
	no	2^nd	19.3	16.3	624.0	15.3	16.7	14.7	13.0	14.0	-
	yes	1^st	25.7	24.7	3349.3	33.3	31.3	36.3	38.7	38.0	-
	yes	2^nd	29.3	27.0	3109.3	30.7	30.3	27.3	30.3	31.7	-
TA 100	no	1^st	201.3	20.0	1786.7	171.3	183.3	207.3	187.3	189.7	-
	no	2^nd	234.3	16.3	1445.3	189.7	188.0	183.0	190.0	206.0	-
	yes	1^st	206.7	24.7	3642.7	198.7	168.0	204.0	195.3	190.0	-
	yes	2^nd	221.0	27.0	3061.3	201.7	199.3	196.0	216.3	214.7	-
Escherichia coli
WP2uvrA	no	1^st	27.3	23.3	282.7	282.7	26.7	26.3	25.7	23.7	-
	no	2^nd	27.7	28.0	378.7	378.7	29.0	28.7	32.7	29.0	-
	yes	1^st	39.7	38.3	261.3	261.3	34.0	45.3	39.3	37.7	-
	yes	2^nd	43.3	40.3	170.7	170.7	36.3	40.7	48.0	43.0	-

Abbreviations:       

Exp.= independent experiment 1 or 2, 1^stexperiment: plate incorporation method, 2^ndexperiment: pre-incubation method

UTC = untreated control

VC = vehicle control

PC = positive control

- = none mutagenic effect

+ = mutagenic effect

Applicant's summary and conclusion

Conclusions:

In conclusion, the test item is not genotoxic in the bacterial reverse gene mutation assay in the presence and absence of mammalian metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria (EU method B.13/14), strains of S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli strain (WP2uvrA) were exposed to Niobium(II) oxide (>99% purity) suspended in DMSO at concentrations of 5.0, 1.0, 0.5, 0.1 and 0.05 mg/plate in the presence and absence of mammalian metabolic activation. Niobium(II) oxide was tested up to the limit dose (5.0 mg/plate). The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable and satisfies the requirement for Test Guideline Directive 67/548/EEC, Annex V, B.13/14 for in vitro mutagenicity (bacterial reverse gene mutation) data.