Hexaammonium heptamolybdate

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:

May 22, 2008 - June 7, 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

study conducted under GLP principles

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: UKEMS Guidelines

Deviations:

not specified

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guidelines

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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

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 / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

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

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

purified water

True negative controls:

no

Controls 2

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Stock solution was formulated in anhydrous analytical grade dimethyl sulphoxide

Controls 3

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Stock solution was formulated in water

Controls 4

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Stock solution was formulated in anhydrous analytical grade dimethyl sulphoxide

Controls 5

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Stock solution was formulated in water

Controls 6

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Stock solution was formulated in anhydrous analytical grade dimethyl sulphoxide

Controls 7

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Stock solution was formulated in anhydrous analytical grade dimethyl sulphoxide

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 results

Test results 1

Species / strain:

S. typhimurium TA 1535

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

Test results 2

Species / strain:

S. typhimurium TA 1537

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

Test results 3

Species / strain:

S. typhimurium TA 98

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

Test results 4

Species / strain:

S. typhimurium 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

Test results 5

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

Applicant's summary and conclusion

Conclusions:

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.