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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 22, 2008 - June 7, 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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
other: UKEMS Guidelines
Deviations:
not specified
Qualifier:
according to
Guideline:
other: ICH Harmonised Tripartite Guidelines
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Sodium Molybdate Dihydrate
- Molecular formula (if other than submission substance): Na2MoO4⋅2H2O
- Molecular weight (if other than submission substance): 241.95 g/mol
- 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 / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
rat liver metabolising system (S-9)
Test concentrations with justification for top dose:
- range-finder experiment: 1.6, 8, 40, 200, 1000, and 5000 µg/plate
- concentrations for experiment 1 were selected based on the results of the range-finder experiment: 1.6, 8, 40, 200, 1000, and 5000 µg/plate
- concentrations for experiment 2: 156.25, 200, 312.5, 625, 1250, 2500, 5000 µg/plate
- concentrations for experiment 2 (TA102 plate-incorporation): 1.6, 8, 40, 200, 1000, and 5000 µg/plate
Vehicle / solvent:
- preparations were made in purified water
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
purified water
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Stock solution was formulated in anhydrous analytical grade dimethyl sulphoxide

Migrated to IUCLID6: 5.0 µg/plate for TA98, no S-9
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Stock solution was formulated in water

Migrated to IUCLID6: 2.0 µg/plate for TA100, TA1535, no S-9
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Stock solution was formulated in anhydrous analytical grade dimethyl sulphoxide

Migrated to IUCLID6: 50.0 µg/plate for TA1537, no S-9
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Stock solution was formulated in water

Migrated to IUCLID6: 0.2 µg/plate for TA102, no S-9
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Stock solution was formulated in anhydrous analytical grade dimethyl sulphoxide. For Experiment 2 pre-incubation treatments, stock solutions were twice the concentration stated. This enabled the volume additions to be reduced to 0.05 mL.

Migrated to IUCL
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene: 5.0 µg/plate for TA100, TA1535, TA1537 and 20.0 µg/plate for TA102, both with S-9
Remarks:
Stock solution was formulated in anhydrous analytical grade dimethyl sulphoxide. For Experiment 2 pre-incubation treatments, stock solutions were twice the concentration stated. This enabled the volume additions to be reduced to 0.05 mL.
Details on test system and experimental conditions:
Preparation of the stock solution:
- Formulation of Sodium molybdate in purified water, stirred where required. Then, the stock solution was membrane filter-sterilised (Pall Acrodisc 32. filter, 0.2 μm pore size) and subsequent dilutions were made using purified water. The solutions were protected from light and used within approximately 5 hours.
- 0.1 mL volume additions of test article solution were used for all treatments

BACTERIA
- the inocula were taken from master plates or vials of frozen cultures, which had been checked for strain characteristics

RANGE-FINDER TEST
- strain TA100 with and without metabolic activation; triplicate plates
- negative (vehicle) and positive controls were included in quintuplicate and triplicate, respectively; with and without metabolic activation
- 0.1 mL bacterial culture, 0.1 mL test article solution or control and 0.5 mL 10% S-9 mix or buffer solution were added to 2.5 mL molten agar at 46±1°C followed by incubation at 37±1°C in the dark for 3 days
- pH measurements were performed: no pH shifts were observed

EXPERIMENTS
- triplicate plates test material
- negative (vehicle) and positive controls were included in quintuplicate and triplicate, respectively; with and without metabolic activation
- the activity of the S-9 mix used in each experiment was confirmed by AAN or B[a]P treatments (again in triplicate) of the strains in the presence of S-9
- 0.1 mL bacterial culture, 0.1 mL test article solution or control and 0.5 mL 10% S-9 mix or buffer solution were added to 2.5 mL molten agar at 46±1°C followed by incubation at 37±1°C in the dark for 3 days (Experiment 1) and for 4 days (experiment 2)
- as the results of Experiment 1 were negative/equivocal, treatments in the presence of S-9 in Experiment 2 included a pre-incubation step: quantities of test article or control solution, bacteria and S-9 mix, were mixed together and incubated for 1 hour at 37±1°C, before the addition of 2.5 mL molten agar
- additional treatments using plate incorporation methodology were performed in strain TA102 in the absence and presence of S-9, to investigate the reproducibility of increases in revertants observed in Experiment 1

COLONY COUNTING
- electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually where confounding factors such as bubbles in the agar or split agar affected the accuracy of the automated counter

-no further significant details stated
Evaluation criteria:
The test article was considered to be mutagenic if:
1. Dunnett's test gave a significant response (p ≤ 0.01) which was concentration related
2. the positive responses described above were reproducible.
Individual plate counts from all experiments were recorded separately and the mean and standard deviation of the plate counts for each treatment were determined. Control counts were compared with the accepted normal ranges for our laboratory for numbers of spontaneous revertants on vehicle control plates and numbers of induced revertants on positive control plates. The ranges that are quoted are based on a large volume of historical control data accumulated from experiments where the correct strain and assay functioning are considered to have been confirmed.
Statistics:
Dunnett's test was used to compare the counts at each concentration with the control. The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels (for example toxicity, precipitation or 5000 μg/plate).

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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
Species / strain:
S. typhimurium TA 102
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:
- for more detailed results see tables in "Attachment documents"

EXPERIMENT 1
- no evidence of toxicity was observed
- Statistically significant increases in revertant numbers were observed in strain TA102 in the presence of S-9 at 200 μg/plate following Experiment 1 treatments (1% level using Dunnett’s test). The increases observed are small and there is no evidence of a dose-related response, which would be evident given the narrowed concentration spacing in the pre-incubation treatment. The increases are therefore not considered to be biologically relevant.

EXPERIMENT 2
- narrowed concentration ranges were employed (156.25 – 5000 μg/plate), in order to examine more closely those concentrations of Sodium molybdate approaching the maximum test concentration and therefore considered most likely to provide evidence of any mutagenic activity
- in addition, treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step (it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system)
- following these treatments no evidence of toxicity was observed
- Statistically significant increases in revertant numbers were observed in strain TA102 in the presence of S-9 at 200 μg/plate following Experiment 2 pre-incubation treatments (1% level using Dunnett’s test). In addition, statistically significant increases were observed when the data were analysed at the 1% level using Dunnett’s test in Experiment 2 treatments of strain TA100 in the absence of S-9 at 200 μg/plate and in strain TA1535 in the absence of S-9 at 2500 μg/plate. The increases observed are small and there is no evidence of a dose-related response, which would be evident given the narrowed concentration spacing in the pre-incubation treatment. The increases are therefore not considered to be biologically relevant.

ADDITIONAL TREATMENTS
- additional treatments in strain TA102 were conducted using plate incorporation methodology to determine the reproducibility of increases in revertants observed in Experiment 1
- following these treatments no evidence of toxicity was observed
- no statistically significant increases were observed in revertant numbers

RANGE-FINDING/SCREENING STUDIES:
- no significant changes in pH were observed at the highest concentration formulated (50 mg/mL) as compared to the concurrent vehicle controls. No adjustment for pH was made and no further measurements were considered necessary
- in the range-finder treatments with Sodium molybdate no clear evidence of toxicity was observed (These data were considered to be acceptable for toxicity assessment only, due to the mean vehicle controls in the absence and presence of S-9 being outside the 99% confidence interval with only two of the five replicates in the presence of S-9 being within the 99% reference range. Correct strain functioning could therefore not be confirmed.)

ADDITIONAL INFORMATION
- the test article was completely soluble in the aqueous assay system at all concentrations treated

- no further significant details stated
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

It was concluded that Sodium molybdate did not induce gene mutations by base pair changes or frame shifts in the genome of the five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 μg/plate, in the absence and in the presence of a rat liver metabolic activation system (S-9). Therefore, Sodium molybdate is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.