Molybdic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 22, 2008 - July 04, 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline conform study conducted under GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Draft proposal for a new Guideline 487: In vitro micronucleus test

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

10102-40-6

Cas Number:

10102-40-6

IUPAC Name:

10102-40-6

Constituent 2

Reference substance name:

sodium molybdate dihydrate

IUPAC Name:

sodium molybdate dihydrate

Details on test material:

- Name of test material (as cited in study report): Sodium Molybdate (Sodium Molybdate Dihydrate, CAS No. 10102-40-6)
- Molecular formula (if other than submission substance): Na2MoO4⋅2H2O
- Molecular weight (if other than submission substance): 241.95
- Physical state: solid (white crystalline powder)
- Analytical purity: 99.9% (dihydrate form) based on an analysed Molybdenum content of 39.6%
- Expiration date of the lot/batch: 31 December 2008
- Storage condition of test material: at room temperature (10-30°C) in the dark, dry
- Other: All Sodium Molybdate concentrations in this report are expressed in terms of the anhydrous test article Sodium Molybdate (CAS 7631-95-0 Na2MoO4), which has a molecular weight of 205.97.
- no further significant details stated

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

lymphocytes: mammalian (human)

Details on mammalian cell type (if applicable):

- Blood from two healthy, non-smoking female volunteers was used for each experiment in this study
- the measured cell cycle time of the donors used at Covance falls within the range 13 +/- 1.5 hours
- for each experiment, an appropriate volume of whole blood was drawn from the peripheral circulation into heparinised tubes within two days of culture initiation
- whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 mL of pooled heparinised blood into 8.1 mL HEPES-buffered RPMI medium (Gibco) containing 20% (v/v) heat inactivated foetal calf serum (PAA Laboratories) and 50 μg/mL gentamycin (Sigma-Aldrich), so that the final volume following addition of S-9 mix/KCl and the test article in its chosen vehicle is 10 mL. The mitogen Phytohaemagglutinin (PHA, reagent grade, Gibco) was included in the culture medium at a concentration of approximately 2% of culture to stimulate the lymphocytes to divide. Blood cultures were incubated at 37°C±1°C and rocked continuously.
- For all treatments conducted, blood cultures were incubated in the presence of PHA for approximately 48 hours prior to treatment.

Metabolic activation:

with and without

Metabolic activation system:

mammalian liver post-mitochondrial fraction (S-9)

Test concentrations with justification for top dose:

- range-finder test: 7.472 to 2060 µg/mL ( all treatments)
- main experiment: 340.4 to 2060 µg/mL (treatment 3+21, with and without S9) and 85.11 to 2060 µg/mL (treatment 24+0, without S9)

Vehicle / solvent:

purified water

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

sterile purified water

Controls 2

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

dissolved in purified water immediately prior to use. For pulse treatments, MMC was used as the positive control. Migrated to IUCLID6: 0.60 and 0.80 µg/mL, without S9

Controls 3

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

CPA was dissolved in sterile anhydrous analytical grade dimethyl sulphoxide (DMSO), frozen (-80°C nominal) and thawed immediately prior to use. Migrated to IUCLID6: 6.25 and12.5 µg/mL, with S9

Controls 4

Positive controls:

yes

Positive control substance:

other: Vinblastine: 0.010, 0.020 and 0.030 µg/mL, without S9

Remarks:

dissolved in purified water immediately prior to use; for the continuous 24+0 hour -S-9 treatment, VIN was used as the positive control.

Details on test system and experimental conditions:

STOCK SOLUTION
- stock solutions were prepared by formulating Sodium Molybdate in purified water with the aid of inversion, where required, to give 20.60 mg/mL. The stock solutions were membrane filter-sterilised (Pall Acrodisc 32, 0.2 μm pore size) and subsequent dilutions made using purified water. They were used within approximately 4 hours.

PRELIMINARY EXPERIMENTS
- A preliminary solubility assessment was performed: a maximum concentration of 2060 μg/mL was selected for the cytotoxicity Range-Finder Experiment, in order that treatments were performed up to 10 mM, the maximum recommended concentration for chromosome aberration studies according to current regulatory guidelines. Concentrations for the Main Experiment were selected based on the results of this cytotoxicity Range- Finder Experiment.
- As part of the preliminary solubility assessment, a stock solution was prepared in water at the maximum concentration required for testing in the study, 20.60 mg/mL. The pH value and osmolality of the stock solution, and of a 10-fold dilution of this solution in culture medium, were measured.

RANGE-FINDER EXPERIMENT (Cytotoxicity)
- Immediately prior to treatment, all cultures designated for continuous treatment had 0.1 mL of culture medium removed to give a final pre-treatment volume of 8.4 mL
- S-9 mix or KCl (0.5 mL per culture) was added appropriately. Cultures were treated with the test article or controls (1 mL per culture). Positive control treatments were not included.
- Cytochalasin B (Sigma), formulated in DMSO was added directly (0.1 mL/culture) to all continuous 24+0 –S-9 cultures at the time of treatment.
- Cultures were incubated at 37°C ± 1°C for the designated exposure time.
- Measurements of pH and osmolality were taken on the stock solution (prior to treatment) and a 10-fold dilution of the stock solution in culture medium (at the time of treatment and post-treatment i.e. upon termination of the treatment) and on post-treatment incubation medium (at either 3 or 24 hours)
- At the time of harvesting, samples were taken for quantification of Caspase activity and hence apoptosis (with Caspase-Glo 3/7 reagent)

MAIN EXPERIMENT
- all cultures designated for positive control treatment had 0.9 mL (pulse treatments) or 0.8 mL (continuous treatments) culture medium added to give final pre-treatment volumes of 9.4 mL or 9.3 mL respectively
- all cultures designated for continuous vehicle or test article treatment had 0.1 mL culture medium removed to give a final pre-treatment volume of 8.4 mL
- S-9 mix or KCl (0.5 mL per culture) was added appropriately
- Cultures were treated with the test article or controls (1 mL per culture or 0.1 mL for positive control cultures)
- The final culture volume was 10 mL. Cultures were incubated at 37°C ± 1°C for the designated exposure time
- Cytochalasin B, formulated in DMSO was added directly (0.1 mL/culture) to all continuous 24+0 –S-9 cultures at the time of treatment. Cultures were incubated at 37°C ± 1°C for the designated exposure time
- for treatment scheme see table 1 (below)
- For removal of the test chemical, cells were pelleted (approximately 300 g, 10 minutes), washed twice with 0.9% sodium chloride (Baxter) (pre-warmed to approximately 37°C), and resuspended in fresh pre-warmed medium containing foetal calf serum and gentamycin. At the appropriate times, Cytochalasin B formulated in DMSO, was added to post wash-off culture medium to give a final concentration of 6 μg/mL per culture.
- Harvesting: at the defined sampling time, cultures were centrifuged at approximately 300 g for 10 minutes, the supernatant removed and discarded and cells resuspended in 4 mL (hypotonic) 0.075 M KCl (Fisher) at 37°C for 4 minutes to allow cell swelling to occur. Cells were then fixed by dropping the KCl suspension into fresh, cold methanol/glacial acetic acid (3:1, v/v [BDH ; Merck]). The fixative was changed by centrifugation (approximately 300 g, 10 minutes) and resuspension. This procedure was repeated as necessary (centrifuging at approximately 1250 g, 2-3 minutes) until the cell pellets were clean. Slides were prepared from the lymphocytes


METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk


DURATION
- Preincubation period:
- Exposure duration:
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):


SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):


NUMBER OF REPLICATIONS:


NUMBER OF CELLS EVALUATED:


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:


OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:


OTHER:

Evaluation criteria:

For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed
3. A concentration-related increase in the proportion of MNBN cells was observed.
After completion of scoring and decoding of slides, the numbers of binucleate cells with micronuclei (MNBN cells) in each culture were obtained. The proportions of MNBN cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test. The proportion of MNBN cells for each treatment condition were compared with the proportion in negative controls by using Fisher's exact test. Probability values of p ≤ 0.05 were accepted as significant. Additionally, the number of micronuclei per binucleate cell were obtained and recorded.

Results and discussion

Test results

Species / strain:

lymphocytes: mammalian (human)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Details on results are shown in the tables in the "Attached background material / Attached documents", see below.
Appenices are included in "Attached background material / Attached documents", see below.

MAIN EXPERIMENTS
- The Main Experiment data presented in this report for the continuous 24+0 hour treatment in the absence of S-9 are from a repeat experiment (trial 2). The data from trial 1 were rejected as the MNBN cell frequencies in the vehicle control cultures were unacceptably high and also due to heterogeneity observed between the replicate cultures at two of the test article concentrations analysed.
- Small, statistically significant increases were observed at the highest concentrations analysed following 24+0 hour and 3+21 hour treatments in the absence of S-9. As these increases were small and not reproduced between replicate cultures, they were considered of questionable biological importance. No such increases were observed following 3+21 hour treatment in the presence of S-9.

RANGE-FINDING/SCREENING STUDIES:
- No significant changes in osmolality or pH were observed at the highest concentration tested (2060 μg/mL) as compared to the concurrent vehicle controls.

COMPARISON WITH HISTORICAL CONTROL DATA:


ADDITIONAL INFORMATION ON CYTOTOXICITY:

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Validity of study

1) The binomial dispersion test demonstrated acceptable heterogeneity (in terms of MNBN cell frequency) between replicate cultures for the majority of treatments (Appendix 2). For the 24+0 hour treatment in the absence of S-9, statistically significant heterogeneity was observed between replicates at the intermediate and highest concentrations analysed (1021 and 1191 μg/mL). At 1021 μg/mL both cultures exhibited MNBN cell frequencies that were within

current historical vehicle control ranges. At 1191 μg/mL a single culture exceeded the normal range. Since this increase was observed in only a single culture, this effect was considered of questionable biological importance. This is not considered to have any negative impact upon the validity of the study in any way.

2) The frequency of MNBN cells in vehicle controls fell within the current historical vehicle control range (Appendix 3)

3) The positive control chemicals induced statistically significant increases in the proportion of MNBN cells (Appendix 1)

4) A minimum of 50% of cells had gone through at least one cell division (as measured by binucleate+ multinucleate cell counts) in negative control cultures at the time of harvest (Tables 14-16).

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

No substantial or reproducible concentration dependant increases in MNBN cell frequency were observed in this study. It is concluded that Sodium Molybdate did not induce micronuclei in cultured human peripheral blood lymphocytes following pulse 3+21 hour treatments in both the
absence and presence of S-9 and following 24 hour treatment in the absence of S-9. Concentrations were tested either up to 10 mM, an acceptable maximum concentration for in vitro chromosome aberration studies according to current regulatory guidelines, or up to the limit of cytotoxicity.