MoVNbTe mixed oxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 August 2021 to 20 December 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted on 29th July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

niobium trivanadium decamolybdenum tellurium dotetracontaoxide

EC Number:

888-364-4

Cas Number:

146569-48-4

Molecular formula:

Mo10V3Nb1Te1O42

IUPAC Name:

niobium trivanadium decamolybdenum tellurium dotetracontaoxide

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
-Batch No.of test material:EX.14402.600

- Expiration date of the lot/batch:No change of properties know over time (endless)
[Unlimited shelf-life (no changes of properties over time known)]
- Purity test date:87.6 %

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29ºC)
- Solubility of test substance in the vehicle: Distilled water
- Reactivity of the test substance with the vehicle of the cell culture medium: NA

Method

Target gene:

HPRT

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

AA8

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells:American Type Culture Collection (ATCC)
- Cell doubling time:Approximately 12 hours


MEDIA USED
Type and identity of media including CO2 concentration if applicable: Alpha MEM CULTURE
MEDIA with 10% FBS and 1% penstrep and 5±1% CO2
- Properly maintained: [yes]
- Periodically checked for Mycoplasma contamination: [yes]
- Doubling time model chromosome number cells were checked.

Metabolic activation:

with and without

Metabolic activation system:

S9 homogenate

Test concentrations with justification for top dose:

0.125, 0.25, 0.5 and 1 mg/mL.
• Since the test item did not precipitate at 0.03125, 0.0625, 0.125, 0.25 and 0.5 mg/mL, moderate precipitation was observed at 1 mg/mL and heavy precipitations was observed at 2 mg/mL. Hence, 1 mg/mL was considered as the highest concentration in the initial cytotoxicity test.
• At a concentration of 1 mg/mL, the Relative Survival was greater than 10%. Therefore 1 mg/mL was selected as the highest concentration for testing in gene mutation test.

• Based on the results of initial cytotoxicity test, four concentrations i.e. 0.125, 0.25, 0.5 and 1 mg/mL were selected for gene mutation test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: found soluble in distilled water at 200 mg/mL

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 2×107METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 2×107

DURATION
- Exposure duration: 3 hours and 1 minute
- Expression time (cells in growth medium): 8 days for mutant phenotype expression
- Selection time (if incubation with a selection agent):7 days

SELECTION AGENT (mutation assays):10 μM of 6-Thioguanine
STAIN : 5% giemsa

NUMBER OF REPLICATIONS:5

METHODS STAINING TECHNIQUE USED: Post incubation period, medium from each culture flask
was aspirated and stained with 5% Giemsa stain. Number of colonies formed was counted manually.
DETERMINATION OF CYTOTOXICITY
- Method:cloning efficiency

Rationale for test conditions:

Rationale for test conditions
Acceptance of a test is based on the following criteria:
•The concurrent vehicle control is considered acceptable for addition to the laboratory historical vehicle control database as described in OECD guidelines for testing of chemicals, No. 476.
•Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative/vehicle control.
•Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results.
•Adequate number of cells and concentrations are analysable (according to OECD guidelines for testing of chemicals, No. 476).
•The criteria for the selection of top concentration are consistent with those described in OECD guidelines for testing of chemicals, No. 476.

Evaluation criteria:

Colony counts in selective media and non-selective media

Statistics:

yes, Statistical analysis of the experimental data was carried out using SPSS Statistical package version 22.0.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No change in pH was observed in any of the test concentrations
- Water solubility: yes
- Precipitation: Precipitation test was conducted at 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/mL to determine the ability of the test item to cause precipitation in the medium. 100 µL of test item (3.125, 6.25, 12.5, 25, 50, 100 and 200 mg/mL) was mixed with 10 mL of culture media and incubated at 37±1ºC with 5±1% CO2 for 3 hours and 30 minutes. Post incubation, results were recorded for change in pH and signs of precipitation.

RANGE-FINDING: yes

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Yes
Positive Control- Benzo(a)pyrene and 4- Nitroquinoline N-oxide
With Metabolic Activation
(3 to 6 hours)
[Benzo(a)pyrene] Without Metabolic Activation
(3 to 6 hours)
[4 Nitroquinoline N-oxide]
Mean Data of Mutant Frequency/2x106 Cells 261.94 264.60
Standard
Deviation 27.28 18.52
Margin of Error 17.82 12.10
Upper bound 279.76 276.70
Lower bound 244.12 252.50

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Relative survivality

Any other information on results incl. tables

 

TABLE 1. SUMMARY OF INITIAL CYTOTOXICITY TEST

Set No.	Treatment	Concentration (mg/mL)	Average Colony Count ± SD			Cloning Efficiency (CE)	Adjusted Cloning Efficiency (ACE)	Relative Survival (RS) (%)
Set 1 +S9	Vehicle Control  (distilled water)	-	185.33	±	6.51	0.93	1.14	-
	test item	0.0625	184.00	±	2.00	0.92	1.11	97.37
		0.125	183.33	±	5.51	0.92	1.10	96.49
		0.25	182.00	±	6.00	0.91	1.08	94.74
		0.5	169.67	±	4.51	0.85	0.98	85.96
		1	158.33	±	2.52	0.79	0.79	69.30
Set 2 -S9	Vehicle Control (distilled water)	-	187.00	±	6.08	0.94	1.15	-
	test item	0.0625	185.00	±	4.58	0.93	1.13	98.26
		0.125	183.33	±	3.06	0.92	1.10	95.65
		0.25	182.33	±	5.86	0.91	1.07	93.04
		0.5	173.33	±	6.11	0.87	1.00	86.96
		1	157.00	±	7.55	0.79	0.80	69.57

 +S9: with metabolic activation; -S9: without metabolic activation;

 Adjusted CE = CE × Number of cells at the end of treatment/number of cells at the beginning of treatment.

 RS = Adjusted CE in treated culture/Adjusted CE in the vehicle control × 100.

 CE = Number of colonies/Number of cells plated.

 

TABLE 2. SUMMARY OF PARALLEL CYTOTOXICITY TEST- GENE MUTATION TEST

Set No.	Treatment	Concentration (mg/mL)	Average Colony Count ± SD			Cloning Efficiency (CE)	Adjusted Cloning Efficiency (ACE)	Relative Survival (RS) (%)
Set 1 +S9	Vehicle Control (distilled water)	-	189.67	±	4.16	0.95	1.15	-
	test item	0.125	183.67	±	5.51	0.92	1.09	94.78
		0.25	182.33	±	3.06	0.91	1.07	93.04
		0.5	172.33	±	7.37	0.86	1.00	86.96
		1	157.33	±	9.07	0.79	0.78	67.83
	Benzo(a)pyrene              (Positive Control)	3 µg/mL	181.33	±	6.81	0.91	1.09	94.78
Set 2 -S9	Vehicle Control (distilled water)	-	188.00	±	3.61	0.94	1.19	-
	test item	0.125	187.67	±	1.53	0.94	1.13	94.96
		0.25	186.00	±	3.61	0.93	1.10	92.44
		0.5	176.33	±	5.51	0.88	1.01	84.87
		1	155.33	±	5.03	0.78	0.79	66.39
	4 Nitroquinoline N-oxide (Positive Control)	1 µg/mL	186.67	±	3.06	0.93	1.11	93.28

+S9: with metabolic activation;  -S9: without metabolic activation;   

 *Note: Cloning Efficiency = 200 cells plated for each replicate.

 RS = Adjusted CE in treated culture/Adjusted CE in the vehicle control × 100.

 CE = Number of colonies/Number of cells plated.

 Adjusted CE = CE × Number of cells at the end of treatment/number of cells at the beginning of treatment.

 

TABLE 3.  SUMMARY OF GENE MUTATION TEST

Set No.	Treatment	Concentration (mg/mL)	*Average Colony Count ± SD			Cloning Efficiency in  selective media	Cloning Efficiency in non-selective media*	Total number of Mutant Colonies/ 2×10^6cells	Mutant Frequency/ 2×10^6cells
Set 1 +S9	Vehicle Control (distilled water)	-	188.33	±	3.79	0.0000122	0.94	23	24.47
	test item	0.125	187.33	±	7.02	0.0000122	0.94	23	24.47
		0.25	187.00	±	2.65	0.0000117	0.94	22	23.40
		0.5	185.00	±	5.29	0.0000124	0.93	23	24.73
		1	184.00	±	2.00	0.0000120	0.92	22	23.91
	Benzo(a)pyrene              (Positive Control)	3 µg/mL	186.33	±	2.52	0.0001290	0.93	240	258.06**
Set 2 -S9	Vehicle Control (distilled water)	-	186.33	±	2.08	0.0000124	0.93	23	24.73
	test item	0.125	186.00	±	3.00	0.0000129	0.93	24	25.81
		0.25	185.00	±	3.00	0.0000129	0.93	24	25.81
		0.5	184.33	±	1.53	0.0000125	0.92	23	25.00
		1	183.67	±	3.06	0.0000125	0.92	23	25.00
	4 Nitroquinoline N-oxide (Positive Control)	1 µg/mL	185.67	±	1.15	0.0001328	0.93	247	265.59**

+S9: with metabolic activation; -S9: without metabolic activation; *:Note: Cloning efficiency = 200 cells plated for each replicate.  

 **: Statistically significant (p˂0.05). 

Mutant Frequency = Cloning efficiency of mutant colonies in selective medium/Cloning efficiency in non- selective medium.

Applicant's summary and conclusion

Conclusions:

Based on the results obtained, the test item is considered as non-mutagenic at and up to the concentration of 1 mg/mL, both in the presence and absence of metabolic activation under the tested laboratory conditions.

Executive summary:

The test item was assessed for its potential to induce gene mutations at the Hprt locus of CHO AA8 cells of the Chinese Hamster.

The test item found soluble in distilled water at 200 mg/mL concentration. Post 3 hours of and 30 minutes of incubation, no precipitation was observed at the tested concentrations of 0.03125, 0.0625, 0.125, 0.25, 0.5 mg/mL, moderate precipitation was observed at 1 mg/mL, and heavy precipitation was observed at 2 mg/mL. No change in pH was observed in any of the test concentrations.

On the basis of precipitation results, 1 mg/mL was selected as the highest concentration for the initial cytotoxicity test. Initial cytotoxicity test was conducted at the concentrations of 0.0625, 0.125, 0.25, 0.5 and 1 mg/mL using distilled water as a vehicle in tetra plates/group in the presence and absence of metabolic activation (3 hours and 17 minutes). Cytotoxicity was assessed by determining the Adjusted Cloning Efficiency and Relative Survival in the test. The results of the initial cytotoxicity test indicated that the Relative Survival was greater  than 10% (69.30 % in presence of metabolic activation and 69.57 % in absence of metabolic activation) at 1 mg/mL when compared with the respective vehicle control, both in the presence and absence of metabolic activation. Based on these results 1 mg/mL was selected as highest concentration for gene mutation test.

The gene mutation test was conducted at the concentrations 0.125, 0.25, 0.5 and 1mg/mL using distilled water as a vehicle in four plates/group in the presence and absence of metabolic activation (3 hours and 1 minute).

Benzo(a) pyrene and 4 Nitroquinoline N-oxide were used as positive controls for the gene mutation test.Cytotoxicity as Relative Survival was 67.83 % in presence of metabolic activation and66.39 % in absence of metabolic activationat the highest tested concentration of 1 mg/mL.

There was no statistically significant increase in mutant frequencies at any of the concentrations tested when compared with the vehicle control. Moreover, treatment with the test item resulted in mutant frequencies which fell within acceptable ranges with regard to historical controls.

There was statistically significant increase in mutant frequenciesfor positive controlswhen compared with the vehicle controlin both metabolic activation and absence of metabolic activation.