Titanium zirconium oxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-10-24 till 2017-11-08

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Guideline:

other: The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE) Guidelines of 31 March 2011

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Titanium zirconium oxide

EC Number:

948-208-9

Molecular formula:

TixZryOz (x = 0.985 - 0.995, y = 1-x, z = 1.65 - 1.75)

IUPAC Name:

Titanium zirconium oxide

Test material form:

solid: bulk

Method

Species / strain

Species / strain / cell type:

other: TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate
The dose selection was not adjusted to purity. Highest recommended dose according to OECD Guideline 471.

Vehicle / solvent:

Solvent used: deionized water
Justification for choice of solvent: best suitable solvent, because of its solubility properties and its relative nontoxicity to the bacteria

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar plate incorporation; pre-incubation

DURATION:
- Preincubation period: 60 Minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3 plates for each concentration including the controls

DETERMINATION OF CYTOTOXICITY: Evident as a reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Plate incorporation test only

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST SPECIFIC CONFOUNDING FACTORS
Effects of pH: none
Water solubility: soluble
Precipitation: The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 µg/plate onward. The undissolved particles had no influence on the data recording.
Other confounding effects: none

COMPARISON WIT HISTORICAL CONTROL DATA:
ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in experiment II in strain TA 1535 with S9 mix at 5000 µg/plate only.

Any other information on results incl. tables

Summary of Experiment I

Metabolic Activation	Test Group	Dose Level (per plate)	TA 1535 Revertant Colony Counts (Mean ± SD)	TA 1537 Revertant Colony Counts (Mean ± SD)	TA 98 Revertant Colony Counts (Mean ± SD)	TA 100 Revertant Colony Counts (Mean ± SD)	WP2 uvrA Revertant Colony Counts (Mean ± SD)
Without	Deionised water		9 ± 3	8 ± 1	21 ± 6	159 ± 15	39 ± 9
Activation	Untreated		11 ± 4	9 ± 5	26 ± 6	160 ± 10	33 ± 7
	Test material	3 µg	11 ± 1	10 ± 4	21 ± 6	144 ± 9	44 ± 10
		10 µg	12 ± 4	9 ± 5	22 ± 8	151 ± 13	37 ± 13
		33 µg	12 ± 3	7 ± 2	22 ± 5	138 ± 29	33 ± 6
		100 µg	9 ± 3	8 ± 3	24 ± 7	146 ± 22	42 ± 4
		333 µg	9 ± 2	8 ± 3	27 ± 2	171 ± 11	43 ± 8
		1000 µg	10 ± 4	10 ± 4	24 ± 6	159 ± 16	37 ± 6
		2500 µg	9 ± 2^P	9 ± 4^P	23 ± 3^P	153 ± 5^P	38 ± 7^P
		5000 µg	10 ± 6^P	8 ± 2^P	21 ± 6^P	159 ± 17^P	39 ± 5^P
	NaN3	10 µg	1123 ± 119			1940 ± 23
	4-NOPD	10 µg			310 ± 45
	4-NOPD	50 µg		138 ± 12
	MMS	2.0 µL					819 ± 11
With	Deionised water		11 ± 4	11 ± 4	29 ± 2	144 ± 13	49 ± 6
Activation	Untreated		13 ± 4	12 ± 5	35 ± 12	141 ± 20	50 ± 16
	Test material	3 µg	11 ± 1	12 ± 5	32 ± 12	129 ± 12	48 ± 0
		10 µg	9 ± 3	12 ± 3	25 ± 3	125 ± 9	37 ± 5
		33 µg	12 ± 3	12 ± 3	37 ± 11	138 ± 7	42 ± 9
		100 µg	10 ± 1	13 ± 4	26 ± 3	127 ± 27	40 ± 13
		333 µg	10 ± 4	10 ± 1	32 ± 9	115 ± 18	45 ± 4
		1000 µg	11 ± 2	9 ± 2	32 ± 5	100 ± 19	57 ± 4
		2500 µg	8 ± 5^P	8 ± 2^P	34 ± 4^P	127 ± 8^P	44 ± 9^P
		5000 µg	14 ± 3^P	10 ± 6^P	32 ± 3^P	142 ± 16^P	49 ± 4^P
	2-AA	2.5 µg	466 ± 35	138 ± 21	3221 ± 133	3153 ± 170
	2-AA	10.0 µg					371 ± 70

Summary of Experiment II

Metabolic Activation	Test Group	Dose Level (per plate)	TA 1535 Revertant Colony Counts (Mean ± SD)	TA 1537 Revertant Colony Counts (Mean ± SD)	TA 98 Revertant Colony Counts (Mean ± SD)	TA 100 Revertant Colony Counts (Mean ± SD)	WP2 uvrA Revertant Colony Counts (Mean ± SD)
Without	Deionised water		12 ± 3	10 ± 1	30 ± 2	183 ± 14	38 ± 9
Activation	Untreated		11 ± 1	11 ± 4	34 ± 2	183 ± 22	42 ± 5
	Test material	33 µg	12 ± 3	11 ± 6	23 ± 7	186 ± 18	40 ± 3
		100 µg	10 ± 3	12 ± 3	31 ± 4	176 ± 15	47 ± 4
		333 µg	11 ± 1	11 ± 4	24 ± 1	200 ± 9	45 ± 8
		1000 µg	10 ± 5	11 ± 2	29 ± 8	185 ± 12	34 ± 4
		2500 µg	9 ± 2^P	8 ± 5^P	24 ± 8^P	177 ± 23^P	40 ± 5^P
		5000 µg	7 ± 3^P	7 ± 3^P	34 ± 1^P	200 ± 17^P	46 ± 4^P
	NaN3	10 µg	1047 ± 105			1940 ± 87
	4-NOPD	10 µg			375 ± 46
	4-NOPD	50 µg		134 ± 7
	MMS	2.0 µL					650 ± 52
With	Deionised water		11 ± 5	13 ± 3	32 ± 8	186 ± 10	59 ± 3
Activation	Untreated		12 ± 2	17 ± 7	40 ± 7	156 ± 8	56 ± 11
	Test material	33 µg	11 ± 4	15 ± 4	36 ± 8	198 ± 18	52 ± 6
		100 µg	11 ± 4	12 ± 4	38 ± 4	176 ± 14	52 ± 2
		333 µg	12 ± 3	12 ± 3	33 ± 4	175 ± 9	54 ± 11
		1000 µg	10 ± 1	16 ± 4	40 ± 1	195 ± 16	60 ± 7
		2500 µg	12 ± 3^P	11 ± 1^P	36 ± 8^P	182 ± 17^P	49 ± 2^P
		5000 µg	3 ± 2^P	12 ± 1^P	35 ± 4^P	195 ± 18^P	50 ± 8^P
	2-AA	2.5 µg	361 ± 50	134 ± 7	4010 ± 761	1542 ± 202
	2-AA	10.0 µg					508 ± 22

Key to Plate Postfix Codes:              

P: Precipitate

Key to positive controls:

NaN3: sodium azide

4 -NOPD: 4 -nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

2-AA: 2 -aminoanthracene

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, Titanium zirconium oxide is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

The test item Titanium zirconium oxide was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) usingSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and theEscherichia colistrain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:       3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:                                33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 µg/plate onward. The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in experiment II in strain TA 1535 with S9 mix at5000 µg/plate only.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Titanium zirconium oxide at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease in induced revertant colonies.