Hexaammonium heptamolybdate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 2020 - February 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2016-07-29

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Disodium molybdate

EC Number:

231-551-7

EC Name:

Disodium molybdate

Cas Number:

7631-95-0

Molecular formula:

Na2MoO4

IUPAC Name:

231-551-7

Test material form:

solid: particulate/powder

Details on test material:

- Physical apperance: white crystalline powder
- Stability: at least until February 2021

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: controlled room temperature (18 - 24°C)

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The Sprague Dawley rat was chosen as the animal model for this study as it is an accepted rodent species for nonclinical toxicity testing by regulatory agencies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Raleigh, NC; USA)
- Age at study initiation: 7 - 8 weeks
- Weight at study initiation: 172 - 265 g
- Assigned to test groups randomly: yes, under following basis: stratified randomisation
- Housing: single housing in solid-bottom cages containing appropriate bedding material (Bed-O-Cobs® or other suitable material)
- Diet: pellet diet (PMI Nutrition International, LLC Certified Rodent LabDiet 5002); ad libitum
- Water: tap water (treated by reverse osmosis and ultraviolet irradiation); ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 26°C
- Humidity: 30 - 70%
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: deionised water

Details on exposure:

Preparation Details:
Dosing formulations were prepared daily at appropriate concentrations to meet dose level requirements. The prepared formulations were not adjusted for purity. The dose formulations were stirred continuously prior to and during dosing. Formulation pH was measured and recorded.

Dose preparation analysis:
Dose formulation samples were collected for analysis of the concentrations of molybdenum. Analyses were performed by inductively coupled plasma mass spectrometry (ICP-MS) using a validated analytical procedure.
Stability analyses performed previously in conjunction with a Range-Finder-Study for a 2-Generation-Reproductive Toxicity Study with the same test item demonstrated that the test substance is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study (Charles River Laboratories, Inc., Horsham, PA 18044, United States, Study No. 20062700). A separate stability analysis was not performed for this study.

Administration of Test Materials:
Test substance formulations and vehicle control were administered once daily, for 2 consecutive days via oral gavage. Each dose was administered via a syringe equipped with an attached gavage cannula.

Duration of treatment / exposure:

two days

Frequency of treatment:

once daily for two consecutive days

Post exposure period:

18 - 24 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

200 mg/kg bw/day

Remarks:

Equivalent dose level: 80 mg Mo/kg bw/day)

Doses / concentrations 2

Dose / conc.:

600 mg/kg bw/day

Remarks:

Equivalent dose level: 240 mg Mo/kg bw/day)

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

Equivalent dose level: 800 mg Mo/kg bw/day). This is the limit dose for this study type.

No. of animals per sex per dose:

6 rats per sex per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

none

Examinations

Tissues and cell types examined:

polychromatic erythrocytes from femoral bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Based on no mortality observed, minimal clinical observations, and decreased body weight/gains across all groups during the Range-Finding Phase, dose levels of 0 (vehicle), 200, 600, and the limit dose of 2000 mg/kg/day were selected for the Definitive Phases by the Sponsor and Study Director.

TREATMENT AND SAMPLING TIMES:
Bone marrow was collected from all animals in each group at the time of euthanasia from the right femur of animals euthanized by inhalation of carbon dioxide. Bone marrow collections were performed approximately 18 to 24 hours following the last dose administration.

DETAILS OF SLIDE PREPARATION:
Bone marrow was aspirated or flushed 2 to 3 times from the right femur into a centrifuge tube using a syringe containing heat inactivated foetal bovine serum (HI FBS). The bone marrow was centrifuged and all but approximately 0.25 mL (or a volume approximately twice that of the cell pellet) of HI FBS was decanted, and the pellet was resuspended in the remaining HI FBS. Bone marrow smears were prepared by placing approximately 1 drop of cell suspension onto a minimum of 4 appropriately labelled, clean microscope slides. Each slide was coded so that the treatment group would not be revealed during subsequent analysis. The slides were air dried, fixed in 100% methanol for approximately 20 minutes, and allowed to air dry a second time. The slides for Definitive Phase II were stored and shipped at ambient temperature to Charles River Skokie for analysis.

METHOD OF ANALYSIS:
Prior to analysis, the coded slides were stained with acridine orange staining solution (Hayashi et al., 1983)*. Slides will be stained for 1 to 3 minutes in AO and then rinsed twice in PBS for a total duration of 2 or 3 minutes. In the event the stain on the slides is too bright, slides may be rinsed in PBS. When a slide appears to be not bright enough, slides may be re-stained. When not in use, slides will be stored protected from light (i.e., in slide boxes). Finally, slides will be wet mounted with a coverslip, using PBS as a mounting medium prior to being read.
Slides from all surviving animals in each group will be analysed. All microscopic analyses will be performed on blinded slides so that the examiner does not know to which treatment group a slide belongs. The number of polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs) will be simultaneously tallied in each field until 500 total erythrocytes (TEs; or NCEs + PCEs) per animal have been scored by sequentially progressing one field at a time. NCEs appear as anucleate cells with a cytoplasm that stains dark, greyish-green. PCEs are also anucleate cells that are generally larger than NCEs with a cytoplasm that stains bright orange. The total number of PCEs and NCEs will be recorded and the ratio of PCE/TE determined. At least 4000 PCEs per animal, when available, will be scored for the presence of micronuclei (MN) to yield the percentage of MN-PCEs (%MN-PCEs). MN stain bright yellow or yellow/green and are generally round or almond shaped within the cytoplasm of the erythrocytes. MN have sharp borders and are generally between 1/20th and 1/5th the size of a PCE. Cells are scored as micronucleated whether they have 1 or more MN, which can sometimes occur.
The number of PCEs, NCEs, and MN-PCEs will be recorded manually. The %MN-PCEs and the ratio of PCE/TE ratio will be calculated for each animal. Group means and standard deviations will be presented.

OTHER:
- Bioanalytical sample collection: First blood samples were obtained from all animals by venepuncture from a jugular vein within two to four hours following the last dose. Approximately 0.5 mL was collected without anaesthesia. Also, prior to necropsy 0.5 mL blood was collected from the retro-orbital sinus of animals anaesthetised with isoflurane.
Plasma samples were mixed gently and centrifuged as within 2 hours of collection. For serum, samples were collected into serum separator tubes and allowed to clot at ambient temperature before centrifugation. The samples were centrifuged and the resultant plasma or serum was separated, transferred to duplicate uniquely labelled polypropylene tubes, and stored frozen in a freezer set to maintain a target of -20°C. The samples to be analysed were shipped on dry ice via overnight courier to the Veterinary Diagnostic Laboratory at Michigan State University.
The samples were analysed for concentration of molybdenum, copper, sulfur, zinc, and iron using an inductively coupled plasma mass spectrometry (ICP-MS) procedure.
- Mortality: At least twice daily (except on days of receipt and euthanasia: at least once daily), beginning upon arrival through termination/release.
- Cageside observations: At the time of dosing and 1–2 hours postdosing.
- Detailed clinical observations: On Days 1 and 2 (prior to dosing). On the day of the scheduled
euthanasia.
- Individual body weights: On Days 1 and 2 (prior to dosing). On the day of the scheduled euthanasia.
- Necropsy: A macroscopic examination of the GI tract and liver was performed and any observations were noted.
- Tissue collection and preservation: Representative samples of selected tissues [saline buffer flushed bone marrow, serum, plasma, liver (both lobes), kidney (both kidneys), and testes or ovaries] were collected from all animals. Following tissue collections, bone marrow, testes, ovaries, 1 liver lobe, and the left kidney were placed into unique tubes and frozen on dry ice until stored in a freezer set to maintain a target temperature of -70°C. The remaining liver lobe and the right kidney were retained in 10% neutral buffered formalin for possible future histopathological evaluation. The frozen samples of kidney and liver lobe were shipped on dry ice to the Veterinary Diagnostic Laboratory at Michigan State University for elemental analysis. The remaining liver lobe and kidney preserved in 10% neutral buffered formalin for possible future histopathological evaluation and frozen testis, ovaries, and saline flushed bone marrow for elemental analysis will be retained.

*References:
Hayashi M, Sofuni T, Ishidate, M Jr. An application of acridine orange fluorescent staining to the micronucleus test. Mutat Res. 1983;120(4):241-247.

Evaluation criteria:

- The test substance will be considered to be clearly positive for induction of MN if:
a) At least one treatment group exhibits a statistically significant increase in % MN-PCEs as compared with the concurrent negative control (p ≤ 0.05); and
b) The increase is dose-related (p ≤ 0.05); and
c) Any increase is outside the 95% control interval of the historical negative control data.

- The test substance will be considered to be clearly negative for induction of MN if:
a) No treatment group exhibits a statistically significant increase in % MN-PCEs as compared with the concurrent negative control; and
b) There is no dose-related increase in %MN-PCEs; and
c) All results are within the 95% control interval of the historical negative control data; and
d) Bone marrow exposure to the test substance has been demonstrated to occur.

Statistics:

The percentage of MN-PCEs and PCEs:TE ratios for the test substance-treated and vehicle control group (Group 9) were subjected to a parametric one-way to determine intergroup differences. If the ANOVA revealed statistically significant (p ≤ 0.05) intergroup variance, Dunnett’s test was used to compare the test substance-treated groups to the vehicle control group. The Cochran Armitage test was used for the determination of dose-response trends. In addition, the positive and vehicle control groups were compared using a separate parametric one-way ANOVA. Statistical significance was assessed at a 95% confidence level (p ≤ 0.05). MN-PCE frequencies were analysed using a 1-tailed test; PCE:TE ratios were analysed using a 2-tailed test.
For body weight analyses, Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

other: The study was conducted up to the limit does of 2000 mg SMD/kg/day (according to OECD TG 474 study). Plasma and serum data confirm that the test item reached the bone marrow.

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Mortality: All animals survived until scheduled euthanasia.
- Clinical observations: Test substance-related clinical observations of activity decreased, and erect fur were noted in the 1000 and 2000 mg SMD/kg/day group males and females. In addition, hunched posture was noted for the 2000 mg SMD/kg/day group males.
- Body weight/gains: Test substance-related decreased body weight/gains were more prominent in the 1000 and 2000 mg SMD/kg/day group males and females as compared to the 500 mg SMD/kg/day group.

RESULTS OF DEFINITIVE STUDY:
- Mortality: One male from the 2000 mg SMD/kg/day group was found dead on Day 3. There were no macroscopic findings for this animal at necropsy. All remaining animals survived until the scheduled necropsy.
- Clinical observations: Test substance-related clinical observations of dehydration suspected (slight or moderate), hunched posture, and erected fur were noted for the 2000 mg SMD/kg/day group males and females. In addition, the 2000 mg SMD/kg/day group males were noted with activity decreased on Day 2 and the 2000 mg SMD/kg/day group females were noted as thin on Day 3.
- Body weight/gains: Test substance-related decreased body weights were noted in the 600 and 2000 mg SMD/kg/day group males and females on Day 2 and Day 3, often reaching statistical significance. In addition, statistically significant test substance-related decreased body weight gains or body weight losses were noted for the 600 and/or 2000 mg SMD/kg/day group males and/or females on Days 1–2 and 2–3.
- Gross pathology: Test substance-related observations of green contents in the large intestine, cecum, and stomach were noted in the 2000 mg SMD/kg/day group males and females. Test substance-related observations of pale liver were noted in the 2000 mg SMD/kg/day group males and females.
- Exposure to target tissue: Assessment of the plasma and serum at 2-4 hours after the second dose indicated a high level of molybdenum demonstrating exposure to the bone marrow.
- Micronucleus evaluation: The test substance did not produce statistically significant or dose-dependent increases in the %MN PCEs in male or female rats at any dose level as compared to the vehicle controls. No bone marrow cytotoxicity (decreases in PCE:TE ratio) was noted in any male or female rats at any dose level. A dose-dependent increase in cytotoxicity was observed for male rats. The test substance was negative for clastogenic activity and/or disruption of the mitotic apparatus under the conditions of this assay.

Applicant's summary and conclusion

Conclusions:

The objective of this study was to assess the potential toxicity of sodium molybdate dihydrate to induce micronuclei in polychromatic erythrocytes in rat bone marrow following 2 consecutive days of treatment when given by oral gavage. Plasma and serum data confirm that the test item reached the bone marrow.

It is concluded that administration of sodium molybdate dihydrate by once daily oral gavage to Crl:CD(SD) rats at dose levels of 200, 600, and 2000 mg SMD/kg/day (the limit dose for an OECD TG 474 study) for 2 consecutive days resulted in a negative response for the induction of bone marrow micronuclei at dosage levels up to 2000 mg SMD/kg/day. The limit dose of 2000 mg SMD/kg/day resulted in the early death of 1 male rat.