Molybdenum zinc tetraoxide

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:

14 August 2018 - 23 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

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:

Molybdenum zinc tetraoxide

EC Number:

237-377-8

EC Name:

Molybdenum zinc tetraoxide

Cas Number:

13767-32-3

Molecular formula:

MoO4Zn

IUPAC Name:

molybdenum(6+) zinc(2+) tetraoxidandiide

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA97, TA98, TA100, TA102, TA1535

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix (rat liver S9)

Test concentrations with justification for top dose:

First experiment: 5000 μg/plate, 1500 μg/plate, 500 μg/plate, 150 μg/plate and 50 μg/plate.
Second experiment: 5000 μg/plate, 2500 μg/plate, 1250 μg/plate, 625 μg/plate, 313 μg/plate and 156 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: In a non-GLP pre-test, the solubility of the test item was tested in demineralized water, di-methyl sulfoxide (DMSO), acetone and tetra hydrofurane. The solid test item was not sufficiently soluble in all solvents. Based on the results of the non-GLP pre-test, acetone was chosen as vehicle, because this vehicle showed a pipetable suspension it does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.

Controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO, Demineralised water and Acetone

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro-1,2-phenylene Diamine (20 µg in DMSO) - TA97a, TA98, TA102 (-S9) // 2-Amino-Anthracene (1 µg in DMSO) - TA97, TA100, TA102 and TA1535 (S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
First experiment: Plate incorporation method.
Second experiment: Pre-incubation method.

Per bacteria strain and concentration, three plates with and three plates without metabolic activation (-S9) were used. For the top agar 100 mL agar basis was melted in a microwave oven, 10 mL of the histidinebiotin-solution 0.5 mM was added, then the mixture was placed in the water bath at 43 ±1 °C. The following materials were gently vortexed in a test tube and poured onto the selective agar plates: 100 μL test suspension at each dose level, solvent (negative control) or reference
mutagen solution (positive control); 500 μL S9 mix; 100 μL bacteria suspension; 2000 μL overlay agar. In the second experiment, after the pre-incubation for 20 minutes, 2000 μL top agar was added. The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37 ±1 °C for 48 h.

DURATION
- Preincubation period: 20 min (second experiment).
- Exposure duration: Incubation for 48 hours at 37 ± 1°C.

NUMBER OF REPLICATIONS: 3

EVALUATION:
The colonies were counted visually and the numbers were recorded. The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item suspensions and the positive controls. Additionally, the absolute number of revertants (mean revertants less mean spontaneous revertants) was given.

OTHER:
Genotype Confirmation:
Genotype confirmation is performed for each batch of lyophilized bacteria before stock cul-ture preparation.

Histidine requirement:
Each strain was streaked on a biotin and a histidine-biotin-plate, using a sterilized wire loop. The plates were incubated for 24 hours at 37 ±1 °C.

Ampicillin/Tetracycline-Resistance (pKM 101, pAQ1)
Each strain was streaked on an ampicillin agar plate and on an ampicillin-tetracycline agar plate. TA1535 was used as control strain, since it is not ampicillin resistant. The plates were incubated for 24 hours at 37 ±1 °C.

UV-sensitivity (uvrB):
Each strain was streaked on a plate, and one half of the plate covered with aluminium foil so that one half of each streak was protected against light. The plates for the strain TA97a, TA100 and TA102 were irradiated for 8 seconds, the plates for the strain TA 98 were irradi-ated for 10 seconds and the plates for the strain TA1535 were irradiated for 6 seconds with a germicidal lamp (254 nm, 30W). Keeping a distance of 33 cm for the strains TA97a, TA102 and TA1535. Keeping a distance of 66 cm for the following strains: TA98, TA100. Incubation for 24 hours at 37 ±1 °C followed.

Crystal violet sensitivity (deep rough/rfa):
For each strain, two plates were used. 0.1 mL of bacteria suspension were mixed with 2 mL Top-Agar and poured on nutrient agar. Sterile paper discs (Ø 9 mm), each soaked with 10 μL of crystal violet solution (0.1%) were placed into the middle of each plate, followed by incubation for 24 hours at 37 ±1°C.

Determination of Titre:
The titre was determined by dilution of the overnight culture using sodium chloride solution and placing 0.1 mL on maximal-soft agar. Incubation for 48 hours at 37 ±1 °C followed. It should give a density of 109 cells/mL (at the least), two replicates with and without metabolic activation.

Toxicity Control
Performed in experiment 1 only analogously to the titre control with the maximum dose of test item on maximal-soft agar, two replicates with and without metabolic activation, incubation for 48 hours at 37 ±1°C.

Evaluation criteria:

A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium, other: TA97, TA98, TA100, TA102, TA1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the first experiment, the test item showed precipitates on the plates in the two highest concentrations (5000 and 1500 μg/plate) in all bacteria strains. In the second experiment, the test item showed precipitates in the two highest concentrations (5000 and 2500 μg/plate) in all bacteria strains.
- Definition of acceptable cells for analysis: All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control.
- Other confounding effects: Concentrations of the test item are stated as nominal concentrations, as suspensions had to be tested due to the poor solubility of the test item. As no complete dissolution was possible, undissolved particles were visible on the plates.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.
- Negative (solvent/vehicle) historical control data: Nearly all determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory.

Any other information on results incl. tables

Mean revertants first experiment:

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	82	88	38	42	102	118	355	331	10	12
	sd	1.0	4.5	1.2	1.7	11.6	20.6	148.7	30.0	1.2	3.0
DMSO	Mean	87	129	40	51	99	111	292	301	10	12
	sd	18.4	23.7	1.2	2.5	15.9	17.5	48.5	19.7	1.5	1.7
Acetone	Mean	130	120	47	48	95	123	471	293	12	11
	sd	22.4	10.7	5.1	2.9	11.0	14.2	128.0	20.1	2.5	2.6
Positive Controls	Mean	613	580	131	295	417	1001	1467	1232	437	146
	sd	68.0	181.6	11.6	34.0	43.9	0.0	95.4	284.4	52.1	40.8
	f(I)	7.05	4.50	3.28	5.78	4.09	9.02	5.02	4.09	43.70	12.17
5000 µg/plate	Mean	97	102	43	40	113	115	384	397	12	11
	sd	19.1	17.1	5.7	4.2	12.9	13.3	26.2	41.1	3.0	0.0
	f(I)	0.75	0.85	0.91	0.83	1.19	0.93	0.82	1.35	1.00	1.00
1500 µg/plate	Mean	82	83	50	39	87	97	393	347	11	11
	sd	11.0	10.0	2.6	5.3	6.6	6.1	4.6	28.4	1.5	0.6
	f(I)	0.63	0.69	1.06	0.81	0.92	0.79	0.83	1.18	0.92	1.00
500 µg/plate	Mean	93	106	45	46	89	113	309	307	12	13
	sd	1.7	31.0	3.6	5.0	7.0	7.0	23.4	23.4	2.1	1.2
	f(I)	0.72	0.88	0.96	0.96	0.94	0.92	0.66	1.05	1.00	1.18
150 µg/plate	Mean	89	77	43	42	107	115	292	295	12	11
	sd	7.0	3.2	11.1	9.0	11.0	14.0	43.3	4.6	2.0	1.5
	f(I)	0.68	0.64	0.91	0.88	1.13	0.93	0.62	1.01	1.00	1.00
50 µg/plate	Mean	94	84	50	46	105	117	287	283	13	13
	sd	17.0	10.1	4.6	6.1	1.2	11.0	28.9	15.1	3.2	2.0
	f(I)	0.72	0.70	1.06	0.96	1.11	0.95	0.61	0.97	1.08	1.18

f(I) = increase factor

1001 colonies per plate means the bacteria growth was too strong for counting.

Mean revertants second experiment:

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	95	91	45	48	101	114	245	260	13	11
	sd	12.1	15.1	4.7	4.2	4.5	17.1	28.4	27.7	2.0	1.5
DMSO	Mean	116	99	47	48	81	101	232	236	12	12
	sd	27.8	25.9	7.1	3.6	5.5	2.3	26.2	28.0	1.2	2.9
Acetone	Mean	99	113	45	51	105	104	237	271	13	11
	sd	10.6	27.0	3.8	3.0	28.6	15.0	2.3	49.4	2.9	2.1
Positive Controls	Mean	635	627	181	177	643	895	1432	1448	232	90
	sd	156.0	60.7	10.1	12.2	34.0	183.0	44.5	28.8	58.1	10.2
	f(I)	5.47	6.33	3.85	3.69	6.37	8.86	6.17	6.14	17.85	7.50
5000 µg/plate	Mean	89	122	48	50	106	107	257	249	11	10
	sd	12.9	7.8	6.4	2.1	14.0	9.9	50.0	47.4	3.2	0.6
	f(I)	0.90	1.08	1.07	0.98	1.01	1.03	1.08	0.92	0.85	0.91
2500 µg/plate	Mean	98	146	50	48	104	107	215	211	10	12
	sd	10.4	2.1	3.8	5.8	2.0	4.2	8.3	18.9	2.0	1.5
	f(I)	0.99	1.29	1.11	0.94	0.99	1.03	0.91	0.78	0.77	1.09
1250 µg/plate	Mean	84	147	50	46	97	124	227	208	10	10
	sd	1.0	27.9	3.1	2.5	6.4	2.5	25.7	8.0	0.6	2.5
	f(I)	0.85	1.30	1.11	0.90	0.92	1.19	0.96	0.77	0.77	0.91
625 µg/plate	Mean	92	125	49	50	99	113	228	261	10	11
	sd	6.4	8.0	6.8	5.3	3.1	16.2	48.7	22.0	2.0	1.7
	f(I)	0.93	1.11	1.09	0.98	0.94	1.09	0.96	0.96	0.77	1.00
312 µg/plate	Mean	87	118	45	48	108	123	223	225	9	11
	sd	4.9	30.4	5.9	6.2	11.5	30.3	24.4	16.2	2.6	2.3
	f(I)	0.88	1.04	1.00	0.94	1.03	1.18	0.94	0.83	0.69	1.00
156 µg/plate	Mean	83	120	53	50	101	114	225	236	9	10
	sd	2.6	14.0	1.5	4.2	8.0	11.7	54.6	50.1	1.7	1.0
	f(I)	0.84	1.06	1.18	0.98	0.96	1.10	0.95	0.87	0.69	0.91

f(I) = increase factor

Historical data of espontaneous revertants:

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Demin. water	Mean	88	95	24	26	93	98	283	302	18	17
	Min	60	63	6	8	51	64	85	67	6	7
	Max	144	138	52	53	147	141	445	587	36	40
	SD	17	17	13	12	15	15	60	72	6	6
	Exp 1	82	88	38	42	102	118	355	331	10	12
	Exp 2	95	91	45	48	101	114	245	260	13	11
DMSO	Mean	89	98	24	25	90	93	285	297	17	17
	Min	58	67	7	8	44	62	79	80	8	6
	Max	139	144	50	50	138	199	465	499	35	37
	SD	17	16	13	12	15	17	61	63	6	6
	Exp 1	87	129	40	51	99	111	292	301	10	12
	Exp 2	116	99	47	48	81	101	232	236	12	12
Acetone	Mean	70	107	21	24	76	84	330	336	17	17
	Min	34	61	6	11	52	54	213	223	10	10
	Max	88	142	52	51	147	140	525	485	36	40
	SD	16	25	14	13	22	21	79	77	6	6
	Exp 1	130	120	47	48	95	123	471	293	12	11
	Exp 2	99	113	45	51	105	104	237	271	13	11
Positive Controls	Mean	539	537	403	138	507	797	1104	1205	266	141
	Min	264	228	77	39	220	273	491	408	55	45
	Max	1165	1181	1001	487	1256	1912	2331	6083	515	712
	SD	166	176	171	105	187	285	397	539	85	84
	Exp 1	613	580	131	295	417	1001	1467	1232	437	146
	Exp 2	635	627	181	177	643	895	1432	1448	232	90

Applicant's summary and conclusion

Conclusions:

It was concluded that the substance is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 up to 5000 μg/plate in the absence and presence of metabolic activation under the experimental conditions in the present study.

Executive summary:

An in-vitro bacterial reverse mutation test was performed according to the OECD Guideline 471 (GLP study). The test item was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535). The test was performed in two experiments up to concentrations of 5000 μg/plate (suspended in acetone) of in the presence and absence of metabolic activation. In the first experiment, the plate incorporation method was used. In the second experiment, the pre-incubation method was used. The test item showed precipitates on the plates at the two highest concentrations (5000 and 1500 μg/plate in the first experiment and 5000 μg/plate and 2500 μg/plate) in all bacteria strains. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation. The results of these experiments showed that the test item caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test item did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.