Dimolybdenum carbide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

2008-05-19 to 2009-01-14

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study. Sodium molybdate used as read across partner.

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

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Sodium Molybdate
- 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):

Whole blood Hepes-buffered RPMI medium:
20% (v/v) heat inactivated foetal calf serum, 50 µg/mL gentamycin, 2% phytohaemagglutinin

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

- range-finder test:
3 hours + 21 hours recovery: 7.472 to 2060 µg/mL with and without S9
24 hours + 0 hours recovery: 7.472 to 2060 µg/mL
- main experiment:
3 hours + 21 hours recovery: 340.4 to 2060 µg/mL with and without S9
24 hours + 0 hours recovery: 85.11 to 2060 µg/mL without S9

Vehicle / solvent:

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

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: vinblastine

Remarks:

Mitomycin C and Vinblastine: without metabolic activaton; cyclophosamide: with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours
- Exposure duration: 3 hours (with and without metabolic activation); 24 hours (without metabolic activation)
- Expression time (cells in growth medium): 21 hours and 0 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

SPINDLE INHIBITOR (cytogenetic assays): cytochalasin B (6 µg/mL)

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 1000 binucleated cells per culture (2000 per concentrations)

DETERMINATION OF CYTOTOXICITY
- Main experiments and dose range finding study: cytotoxicity (%) is expressed as 100 - relative Replication Index
- in the dose range finding study additionally apoptosis was assessed at the time of harvesting. Samples were taken for quantification of Caspase
activity by using the Caspase-Glo assay (Promega, UK)

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.

Statistics:

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: peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

In conclusion, it can be stated that during the study described, Sodium molybdate did not induce structural and/or chromosomal damage in human peripheral blood lymphocytes. Therefore, Sodium molybdate is considered to be non-mutagenic in this in vitro mammalian cell micronucleus test.

Executive summary:

In an in vitro mammalian micronucleus assay (OECD 487), primary human peripheral blood lymphocyte cultures were exposed to Sodium molybdate (99.9%), solved in purified water, at a concentration range of 340.4 to 2060 µg/mL (3 hours treatment, with and without metabolic activation) and of 85.11 to 2060 µg/mL (24 hours treatment, without metabolic activation). Sodium molybdate was tested to the maximum concentration of 0.01 M, equivalent to 2060 µg/mL. The positive controls induced the appropriate response. There was no evidence of induced structural and/or numerical chromosomal damage in comparison to the untreated negative control. Sodium molybdate was used as read-across partner to Dimolybdenum carbide.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 487.