Dipotassium tetraoxomolybdate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD TG 471 (Ames test): negative

OECD TG 487 (in vitro micronucleus test): negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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: Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA
- Suitability of cells: The use of the strains was in accordance with the current scientific recommendations for the conduct of this assay.

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA
- Suitability of cells: The use of the strain was in accordance with the current scientific recommendations for the conduct of this assay.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from phenobarbital and β-naphthoflavone induced rat liver

Test concentrations with justification for top dose:

0, 33, 100, 333, 1000, 2500, 5000 ug/plate

Justification:
In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ultrapure water

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene (2-AA); N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); 4-nitro-o-phenylenediamine (NOPD)

Details on test system and experimental conditions:

METHOD OF APPLICATION: standard plate test and preincubation test

STANDARD PLATE TEST
The experimental procedure of the standard plate test (plate incorporation method) was based on the method of Ames et al.
- Salmonella typhimurium:
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept in a water bath at about 42 - 45°C, and the remaining components were added (test solution or vehicle; fresh bacterial culture; S9 mix or phosphate buffer). After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.
- Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added (test solution or vehicle; fresh bacterial culture; S9 mix or phosphate buffer). After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (trp+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.

PREINCUBATION TEST
The experimental procedure was based on the method described by Yahagi et al. and Matsushima et al.
0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) were incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted
manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed with S9 mix at 5000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed starting at 2500 ug/plate with and without S9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed with S9 mix at 5000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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

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:

No test substance precipitation was found with and without S9 mix.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

yes

Remarks:

In-house non-GLP validation experiments were performed to determine optimal study design using the specified positive controls. To achieve such response the test design was slightly modified comparing the current proposal given in the OECD TG 487.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Blood samples were drawn from healthy non-smoking donors not receiving medication. For this study, blood was collected from a male donor (24 years old) for Experiment I and from a female donor (31 years old) for Experiment II. The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.


MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection. The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

Cytokinesis block (if used):

Cytochalasin B (4 μg/mL)

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

0, 13.0, 22.7, 39.8, 69.6, 122, 213, 373, 653, 1143, 2000 ug/mL

Justification: Dose selection was performed according to the current OECD Guideline for the in vitro micronucleus test. If no precipitate or limiting cytotoxicity is observed, the highest test concentration should correspond to 10 mM, 2 mg/mL or 2 μl/mL, whichever is the lowest.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:deioinized water
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcine

Details on test system and experimental conditions:

PULSE EXPOSURE
About 48 hrs after seeding, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration and each control. The culture medium was replaced with serum-free medium containing the test item. For the treatment with metabolic activation, the culture medium was supplemented with approx. 2.5 % S9 fraction (50 μL S9 mix/mL culture medium). After 4 hrs the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were resuspended in and washed with "saline G" (pH 7.2, containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose •H2O, 192 mg/L Na2HPO4 • 2 H2O and 150 mg/L KH2PO4). The washing procedure was repeated once as described. The cells were resuspended in complete culture medium with 10 % FBS (v/v) and cultured for a 16-hour recovery period. After this period Cytochalasin B (4 μg/mL) was added and the cells were cultured another approximately 20 hours until preparation.

CONTINUOUS EXPOSURE (without S9 mix)
About 48 hrs after seeding, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration and each control. The culture medium was replaced with complete medium (with 10 % FBS) containing the test item. After 20 hours the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were re-suspended in and washed with "saline G". The washing procedure was repeated once as described. After washing the cells were re-suspended in complete culture medium containing 10 % FBS (v/v). Cytochalasin B (4 μg/mL) was added and the cells were cultured another approximately 20 hours until preparation.

PREPARATION OF CELLS
The cultures were harvested by centrifugation 40 hrs after beginning of treatment. The cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were re-suspended in approximately 5 mL "saline G" and spun down once again by centrifugation for 5 minutes. Then the cells were resuspended in 5 mL KCl solution (0.0375 M) and incubated at 37 °C for 20 minutes. 1 mL of ice-cold fixative mixture of methanol and glacial acetic acid (19 parts plus 1 part, respectively) was added to the hypotonic solution and the cells were resuspended carefully. After removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with Giemsa.

EVALUATION OF CYTOTOXICITY AND CYTOGENETIC DAMAGE
Evaluation of the slides was performed using microscopes with 40 x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. The frequency of micronucleated cells was reported as % micronucleated cells. To describe a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.

Statistics:

Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

Key result

Species / strain:

lymphocytes: human

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:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Water solubility: soluble
- Precipitation: no

Experiment 1

	Concentration	Exposure time	Preparation interval	CBPI per 500 cells*	Cytostasis (%)
Without S9 mix	Solvent control	4 hrs	40 hrs	2.12	-
	653	4 hrs	40 hrs	2.10	1.7
	1143	4 hrs	40 hrs	2.09	2.1
	2000	4 hrs	40 hrs	2.08	3.2
With S9 mix	Solvent control	4 hrs	40 hrs	2.08	-
	653	4 hrs	40 hrs	2.08	0.3
	1143	4 hrs	40 hrs	2.09	n.c.
	2000	4 hrs	40 hrs	2.10	n.c.

 

 

Experiment 2

	Concentration	Exposure time	Preparation interval	CBPI per 500 cells*	Cytostasis (%)
Without S9 mix	Solvent control	20 hrs	40 hrs	2.05	-
	653	20 hrs	40 hrs	2.22	n.c.
	1143	20 hrs	40 hrs	2.01	3.0
	2000	20 hrs	40 hrs	1.76	27.8

Only groups evaluated for cytogenetic damage are shown

* Mean value of two cultures

n.d. Not determined

n.c. Not calculated as the CBPI was equal or higher than solvent control value

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

no reliable study addressing in vivo genetic toxicity of test substance available.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Data available on potassium molybdate:

For the test substance itself, an Ames test (according to OECD TG 471 and GLP) as well as an in vitro micronucleus test in human lymphocytes (according to OECD TG 487 and GLP) are available. Apart from these recent highly reliable data, two literature reports on genotoxicity of potassium molybdate exist.

In a bacillus subtilis recombination assay, Nishiokaet al.investigated the mutagenic capacity of several metal salts. Comparing Rec- and Rec+ strains, they determined a potential for genotoxicity for potassium molybdate. However, this study is considered not reliable, since the reporting was incomplete, purity of test material was not stated and the results obtained for other metal salts were inconsistent with other publications.

Ogawaet al.used a sister chromatid exchange assay to determine possible chromosomal damage by potassium molybdate. Human blood cells were treated, however of only one donor and metabolic activation was not specified. Effects were observed, however only at lower concentrations, whereas cytotoxicity was observed at higher concentrations. Apart from the reporting and methodological deficiencies of this study, it cannot be excluded that the effects observed are due to an artifact. Therefore, also this study was considered unreliable and disregarded.

In contrast, the reliable, recently conducted OECD TG 471 and GLP-conform Ames test shows no mutagenic potential for the test substance. Similarly, a recently conducted OECD TG 487 and GLP-conform in vitro micronucleus test in human lymphocytes did not detect clastogenic potential for the test substance.

 

Data available on sodium molybdate (read-across substance):

The following recently conducted guideline-conform, highly reliable genotoxicity tests are available for sodium molybdate:

- an AMES bacterial reverse mutation assay (Beevers, 2009),

- an in vitro micronucleus assay in human lymphocytes (Taylor, 2009), and

- an in vitro gene mutation assay (tk) in mouse lymphoma cells (Lloyd, 2009).

All three tests produced unequivocally negative results, and thus provide strong evidence for an absence of concern for genotoxic effects of molybdenum substances. For justification of read-across, please refer to justification document attached in IUCLID chapter 13.

 

Apart from these high-quality test results, several published studies for sodium molybdate exist, which are of varying quality and extent of documentation. These were subjected to a detailed quality and reliability screening, the outcome of which can be summarized as follows:

(i)           Negative results were obtained in an Ames test, which however from a perspective of formal regulatory compliance is considered incomplete because of the selected tester strains (Kuboet al.2002).

(ii)          Further, negative results were obtained in a Saccharomyces cerevisiae mutation test, however also this study is considered inadequate due to deficiencies with regard to test substance purity and reporting (Singhet al.1983)

(iii)         Ambiguous results were obtained in an E. coli assay by Rossmannet al.(1984), due to inadequate test substance purity, incomplete test design and borderline result, this study was considered not reliable for assessment.

(iv)         An in vitro MNT provided positive results, however several methodological and reporting deficiencies render this study unreliable (Titenko-Hollandet al.1998).

In view of the unequivocally negative results in allin vitrokey studies, the conduct of any furtherin vivotesting would not be required. Already availablein vivostudies for sodium molybdate were subjected to a thorough evaluation and were found to be seriously flawed and of poor quality, and were thus assigned reliability grades of either 3 or 4. In this context it is explicitly noted that set of studies published by Titenko-Holland (1998) have already previously been criticized and disregarded by the Classification and Labelling Committee of the ECB as deficient during discussions on molybdenum trioxide in 2004.

 

Overall conclusion:

The available high-quality data on potassium molybdate and sodium molybdate do not suggest a concern for genotoxicity.

Justification for classification or non-classification

Based on the reliable data available and according to to the criteria for classification and labeling laid down in regulation (EC) 1272/2008 (CLP), the criteria for classification are not met. Therefore, non-classification of the test substance is warranted.