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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-10-07 to 2011-10-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliable without restrictions: Guideline study (OECD 408), conducted and reported in accordance with GLP principles. The study includes additional parameters, such as analysis of oestrous cycles and sperm, as per guideline OECD 416.
Cross-reference
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
modified to include oestrous cycle and sperm analyses as specified in OECD 416.
GLP compliance:
yes
Remarks:
Only analysis of dose formulations,blood & tissues were non-GLP (the lab Michigan State University is non-GLP).However,the laboratory is fully certified under the American Association of Veterinary Laboratory DDiagnosticians (AA VLD) and has a QC program.
Limit test:
no

Test material

Constituent 1
Reference substance name:
10102-40-6
EC Number:
600-158-6
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 dihydrate
- Molecular formula: Na2MoO4.2H2O
- Molecular weight: 241.95
- Physical state: white crystalline powder
- Analytical concentration: 39.5 ± 0.8% Mo (theoretical content is 39.65, so material is > 99% pure)
- Expiration date of the batch: 04-05-2011 (last dosing of animals: 25-01-2011)
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
other: Crl: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: approximately 7 weeks
- Weight at study initiation (mean weight): males: 338.4 g (range: 309.8 - 377.6 g); females: 229.6 g (range: 187.9 - 263.5 g)
- Housing: animals were individually housed in elevated, stainless steel, wire mesh cages. An enrichment device (e.g., a Nylabone®) was provided in each animal’s cage at all times. Previous analysis of Nylabone in the range finder study (please refer to Section 7.5.1 Repeated dose toxicity: oral: s_Hoffman_2011_28 d_gavage and Section 7.5.1 Repeated dose toxicity: oral: s_Hoffman_2011_28 d_dietary administration) showed that it contained no significant amount of molybdenum (<1ng Mo/g).
- Diet (ad libitum): Certified Rodent Diet, No. 2016C (meal) (Harlan Teklad, Madison, Wisconsin); fresh feed was presented weekly during the study except during Week 1 when fresh feed was presented 3 times.
- Water (ad libitum): water (New Jersey-American Water Company, Cherry Hill, New Jersey) via an automated watering system.
- Acclimation period: approximately 2.5 weeks; all animals were examined during the stabilization period to confirm suitability for study.

Currently acceptable practices of good animal husbandry were followed e.g., Guide for the Care and Use of Laboratory Animals; National Academy Press, 1996.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 23°C (daily average range)
- Relative humidity: 34 to 49% (daily average range)
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: Certified Rodent diet, No. 2016C
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): fresh dose formulations were prepared once weekly for the first 4 weeks of the study and then every other week for the rest of the treatment period to approximate as closely as possible the target dose levels in mg/kg bodyweight. Dose formulations were prepared as averaged mixtures for the males and females (based on body weight and feed consumption data from the preceding interval and the molecular weight ratio of the test substance) in each group
- Mixing appropriate amounts with Certified Rodent diet, No. 2016C
- Storage temperature of food: stored at room temperature in tightly sealed bags when not in use. It was confirmed in a range finder study (please refer to Section 7.5.1 Repeated dose toxicity: oral: s_Hoffman_2011_28 d_gavage and Section 7.5.1 Repeated dose toxicity: oral: s_Hoffman_2011_28 d_dietary administration) that dose formulations are stable for at least 5 weeks when prepared and stored at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses to determine homogeneity and concentration of the test substance with carriers were performed. A validated method for molybdenum, copper, zinc and manganese was used.
- Homogeneity and confirmation analysis:
Samples of diet formulations (approximately 50 g in duplicate from the top, middle and bottom from the 1st week’s preparation, and approximately 50 g in duplicate from the middle from each subsequent preparation) for Groups 1 to 4 were collected from each prepared batch after preparation. Dose formulation samples were stored at room temperature in tightly sealed bags. Due to questionable analytical results, the back-up samples from the Week 1 formulations were blind labelled and shipped for analysis. These confirmed the original results.
-Stability: stability for at least 5 weeks of storage was determined for samples generated in a range finder study (please refer to Section 7.5.1 Repeated dose toxicity: oral: s_Hoffman_2011_28 d_gavage and Section 7.5.1 Repeated dose toxicity: oral: s_Hoffman_2011_28 d_dietary administration)

- Method of analysis:
Molybdenum analysis was performed by inductively coupled plasma - mass spectroscopy (ICP-MS).

Feed samples were dried overnight in a 75 degree Celsius oven, and a dry matter ratio was obtained by measuring the moisture lost in drying. Dry feeds were weighed out and digested overnight in a 95 degree Celsius oven in individual sealed vessels with 1 mL nitric acid: 100 mg feed ratio. The resulting solution was diluted with 18 MΩ water to a final mass of 25 g. The concentrated solutions and salts were further diluted with 18 MΩ water to lower the concentration of the analytes in the diluted samples into the calibration range.
Three modes were used to minimize the spectral interferences for the analysis, copper (mass 65), zinc (mass 66) and cobalt (mass 59) were analysed in helium mode. Selenium (mass 78) and iron (mass 56) were analysed in hydrogen mode. Lastly, manganese (mass 55) , and molybdenum (mass 95) were analysed in non-gas mode.

Results:
Analysis confirmed that the preparation procedure used for this study produced homogeneous mixtures under storage conditions used in this study. Analyses conducted during the treatment period confirmed that dose formulations of appropriate concentration were administered. The initial results from the Week 1 preparations were suspect (they varied between 83-121% of the expected results) but analysis of secondary (and blinded samples) showed similar results and thus the initial results were accepted for summary calculations below.
Mean nominal and analytical Mo results, expressed as concentration and percent of nominal (desired) concentrations were as follows:

Nominal Mo concentration:
Group 1 (control group): 0 ppm
Group 2 (5 mg Mo/kg bw/day): 75 ppm
Group 3 (17 mg Mo/kg bw/day): 263 ppm
Group 4 (60 mg Mo/kg bw/day): 896 ppm

Analytical Mo concentration:
Group 1 (control group): 0.9 ppm
Group 2 (5 mg Mo/kg bw/day): 68 ppm
Group 3 (17 mg Mo/kg bw/day): 268 ppm
Group 4 (60 mg Mo/kg bw/day): 907 ppm

Analytical Mo concentration (% of nominal):
Group 1 (control group): not applicable
Group 2 (5 mg Mo/kg bw/day): 91
Group 3 (17 mg Mo/kg bw/day): 102
Group 4 (60 mg Mo/kg bw/day): 101

The Mo concentration in Group 1 samples was considered typical and expected background.
Duration of treatment / exposure:
Test and control groups: 91 (males) or 92 (females) days
Frequency of treatment:
Test and control groups: ad libitum feed presentation.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 5, 17 and 60 mg/kgbw/day
Basis:
actual ingested
No. of animals per sex per dose:
Group 1 (control group): 10 males / 10 females (for sacrifice/necropsy after 90 days) PLUS an extra 10 males and 10 females for a 60 day recovery period.
Group 2 (5 mg Mo/kg bw/day): 10 males / 10 females
Group 3 (17 mg Mo/kg bw/day): 10 males / 10 females
Group 4 (60 mg Mo/kg bw/day): 10 males / 10 females (for sacrifice/necropsy after 90 days) PLUS an extra 10 males and 10 females for a 60 day recovery period.
Control animals:
yes, plain diet
Details on study design:
Dose selection rationale:
- In a study by Pandey and Singh (Pandey and Singh, 2002)*, 50 mg/kg bw sodium molybdate was possibly toxic to the testes and bodyweight when given as an oral bolus dose (probably gavage), 5 days/week for 60 days. In the same study, 30 mg/kg was a LOAEL and 10 mg/kg bw was possibly a NOAEL or might be a LOAEL.
- In a study by Cox et. al. (1960)*, Mo was given as sodium molybdate in two synthetic diets at 500 ppm (about 50 mg/kg bw) for 5-8 weeks. This proved to be toxic with diarrhea and decreased weight gain, and with high liver molybdenum levels and no effect on liver copper stores.
- All the rats died during the first week of a study where they were given 400 mg Mo/kg bw/day in the diet (Nielands et al., 1948)*.
- In a range-finding study (please refer to Section 7.5.1 Repeated dose toxicity: oral: s_Hoffman_2011_28 d_gavage and Section 7.5.1 Repeated dose toxicity: oral: s_Hoffman_2011_28 d_dietary administration), there were no remarkable treatment effects, including no effects on the testes, from oral gavage or dietary dosing at 4 and 20 mg Mo/kg bw/day for 28 days (equivalent to 10 and 50 mg/kg bw/day of sodium molybdate dihydrate).

Therefore, for this study, it was estimated that the low dose (5 mg Mo/kg bw/day) should have been without effect based on the results of a range-finding study (please refer to Section 7.5.1 Repeated dose toxicity: oral: s_Hoffman_2011_28 d_gavage and Section 7.5.1 Repeated dose toxicity: oral: s_Hoffman_2011_28 d_dietary administration) and previous published studies. The selection for the middle dose (17 mg Mo/kg bw/day) was based on the fact that it is logarithmically between the high and low dose. It was expected that some effects at the high dose (60 mg Mo/kg bw/day) would be seen, based on other published studies. In addition, palatability was not expected to be a problem since Arrington et. al. (1965)* did not see a decrease in food consumption in rats given up to 80 mg Mo/kg bw/day in the diet (as sodium molybdate dihydrate) for 6 weeks. Also, 60 mg Mo/kg bw/day represents a dose level which is about 20,000 times higher than typical human dietary intake (205 mcg Mo/day or 3 mcg Mo/kg bw/day) and it was estimated that much higher doses than 60 mg Mo/kg bw/day would result in undesired mortality.

* References:
- Arrington, L.R. et. al. 1965. Molybdenum toxicity in rats and rabbits. Journal of the Florida Academy of Science 28: 129-136.
- Cox, D., et al. 1960. Influence of excess dietary molybdenum on rat and calf liver and heart enzymes. Journal of nutrition. 70: 63-68.
- Neilands. J.B., Strong. F.M., Elvehjem. C.A. 1948. Molybdenum in the nutrition of the rat. J. Biol. Chem. 2: 431-439.
- Pandey R. and Singh S.P. 2002. Effects of molybdenum on fertility of male rats. Department of Zoology, University of Lucknow, UP Biometals. 1: 65-72.

- Post-exposure recovery period in satellite groups: recovery groups were kept on normal untreated diet for 59 days (females) and 60 days (males) prior to termination.
Positive control:
None.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and general condition; during the treatment period, all animals were observed for signs of toxic or pharmacologic effects at least twice daily. These observations were made concurrently with the viability checks.

DETAILED CLINICAL OBSERVATIONS AND NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule: animals were removed from their cages and examined once pretest and weekly during the study period (except examinations were
performed twice during the first week of the recovery period).
- Examinations included observations of skin and fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects, including tremors and convulsions, changes in the level of motor activity, gait and posture, reactivity to handling or sensory stimuli, grip strength, and stereotypies or bizarre behaviour (e.g., self-mutilation, walking backwards) according the Testing Facility SOPs describing detailed physical and behavioural examination.
- Grip strength was measured by allowing the animal to grip an inverted cage and then applying a gentle, horizontal pull on the tail, slowly drawing the animal backward. The grip strength was determined in terms of gripping resistance of the animal to this action.

BODY WEIGHT: Yes
- Time schedule for examinations: animals were weighed twice pretest, weekly during the study and terminally (after fasting). Terminal, fasted body weights were obtained just prior to necropsy.

FOOD CONSUMPTION: Yes
- Feed was available without restriction 7 days/week. Animals were presented with full feeders of known weight. After up to 6 days, feeders were reweighed and the resulting weight was subtracted from the full feeder weight to obtain the grams consumed per animal over the up to 6-day period.
- Food consumption was measured (weighed) during the week prior to treatment initiation (over a 6-day period), at Days 2 and 4 and 7 in the first week of
dosing.
- The amount of food consumed over a 6-day period was used to determine feed concentration calculations for Week 2 and weekly (over a 6-day period) for the first 4 weeks and every other week during the rest of the study.

TEST SUBSTANCE INTAKE: Yes
Calculated from food consumption data and based on nominal dietary concentrations:
Test Substance Intake (mg Mo/kg bw/day) = Food consumed (g/kg bw/day) x concentration of molybdenum in diet (mg Mo/g diet)
The current body weight was used in the calculation.

FOOD CONVERSION EFFICIENCY: Yes
Calculated from weekly body weight and food consumption data:
Food Conversion Efficiency = (body weight gain (g)/ food consumption (g/interval)) x 100

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: animals were examined pretest and at termination of the treatment period. Lids, lacrimal apparatus and conjunctiva were examined visually. The cornea, anterior chamber, lens, iris, vitreous humor, retina and optic disc were examined by indirect ophthalmoscopy.
Mydriacyl 1% was used to induce mydriasis.
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood and anaesthetic used: blood obtained via the jugular vein (unanaesthetized or when necessary lightly anaesthetized with isofluorane) or the orbital sinus (lightly anaesthetized with isofluorane) was used to analyse hematology and coagulation parameters at termination of the treatment period.
- Animals fasted: Yes, prior to blood collection
- How many animals: 10 animals/sex/group
- Parameters checked: haemoglobin concentration, haematocrit, erythrocyte count, platelet count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, red cell distribution width, total leukocyte count, reticulocyte count, differential leukocyte count1, prothrombin time and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood and anaesthetic used: blood obtained via the jugular vein (unanaesthetized or when necessary lightly anaesthetized with isofluorane) or the orbital sinus (lightly anaesthetized with isofluorane) was used to analyse clinical chemistry parameters at termination of the treatment period.
- Animals fasted: Yes, prior to blood collection
- How many animals: 10 animals/sex/group
- Parameters checked: aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, blood urea nitrogen, creatinine, glucose, cholesterol, triglycerides, total protein, albumin, uric acid, total bilirubin, sodium, potassium, chloride, calcium, inorganic phosphorus, globulin and albumin/globulin ratio (calculated value; albumin ÷ globulin)

URINALYSIS: No

BLOOD MOLYBDENUM:
Blood samples were obtained for the determination of serum concentrations of molybdenum (and copper, zinc and manganese).
- Collection intervals: during Week 4 (Days 22 to 25), Week 12 (Days 78 to 81) and during the 1st week of the recovery period (2 days and 7 days after
termination of the treatment period);
- No. of annimals: blood samples were obtained for molybdenum determinations from all surviving animals at each interval. During Weeks 4 and 12, approximately equal numbers of animals per sex per group per day were sampled. Samples were collected at approximately 0900 (±75 minutes) on each day.
- Collection procedures: approximately 0.5 mL of whole blood was obtained from each unanaesthetized animal via the jugular vein. When necessary, animals were lightly anesthetized with isofluorane. Animals were not fasted prior to blood collection. Blood was collected into plastic silicone coated interior (Royal Blue Top) tubes, containing no additive and placed at room temperature in an upright position and allowed to clot for at least 30 minutes.
In Week 12 for all animals scheduled for terminal sacrifice, blood (approximately 0.25 mL) was also collected into lithium heparin tubes and inverted and placed into wet ice.
- Processing, storage and disposition of samples: blood samples collected into plastic silicone coated interior tubes were processed to obtain serum. Serum was separated by centrifugation (for 10 minutes at approximately 3000 rpm, at approximately 2–8°C). Serum (approximately 0.2 mL) was transferred into a cryotube. Tubes were stored frozen at approximately -80°C (±10°C) (within 2 hours after collection of each blood sample).
Blood samples collected into tubes containing lithium heparin were transferred into cryotubes and stored refrigerated at approximately 2 to 8 °C (within 2 hours after collection of each blood sample).
- Sample analysis and reporting: serum samples were analysed with a validated inductively coupled plasma mass spectrometry (ICP-MS) method under non-GLP conditions.
200 µL of each digest, and serum/blood samples, were pipetted and diluted with 5 mL of a solution containing 0.5% EDTA and Triton X-100, 1% ammonia hydroxide, 2% propanol and 20 ppb of scandium, rhodium, indium and bismuth as internal standards. An Agilent 7500ce Inductively Coupled Plasma - Mass Spectrometer (ICP-MS) was used for the analysis. The ICP-MS was tuned to yield a minimum of 5000 cps sensitivity for 1 ppb yttrium (mass 89), less than 1.0% oxide level as determined by the 156/140 mass ratio and less than 2.0% double charged ions as determined by the 70/140 mass ratio. Each element was calibrated using a 4 point linear curve of the analyte: internal standard response ratio.
Three modes were used to minimize the spectral interferences for the analysis, copper (mass 65), zinc (mass 66) and cobalt (mass 59) were analysed in helium mode. Selenium (mass 78) and iron (mass 56) were analysed in hydrogen mode. Lastly, manganese (mass 55) , and molybdenum (mass 95) were analysed in non-gas mode.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Necropsy was performed on up to 10 animals/sex/group after animals had been treated for up to 92 days. Animals were fasted overnight prior to necropsy. Necropsy of the remaining 10 animals/sex/group from Groups 1 and 4 occurred after the animals had been allowed to recover for up to 60 days after termination of the treatment period.
- A necropsy schedule was established to ensure that approximately equal numbers of males and females were examined on each day of necropsy and that examination of animals of both sexes were performed at similar times of the day throughout the necropsy period.
- Exsanguination following carbon dioxide inhalation.
- Complete macroscopic examinations were performed on all animals including examination of the external surface and all orifices; the external surfaces of the brain and spinal cord; the organs and tissues of the cranial, thoracic, abdominal and pelvic cavities and neck; and the remainder of the carcass for the presence of macroscopic morphologic abnormalities.

ORGAN WEIGHTS:
- The following organs were weighed for all animals at the scheduled sacrifice intervals: adrenal glands, brain (medulla, pons, cerebrum and cerebellum), epididymides, heart, kidneys, liver, ovaries, pituitary gland (weighed post-fixation), prostate gland, seminal vesicles, spleen, testes, thymus, thyroid/parathyroid glands (weighed post-fixation), and uterus (body/horns) with cervix.
- Prostate and seminal vesicles were weighed together.
- Prior to weighing, the organs were carefully dissected and properly trimmed to remove adipose and other contiguous tissues in a uniform manner. Organs were weighed as soon as possible after dissection in order to avoid drying. Paired organs were weighed together.

HISTOPATHOLOGY: Yes
- The following tissues were obtained during the necropsies and preserved for all animals: adrenal glands, aorta (thoracic), bone (sternum, distal femur), bone marrow (sternum, distal femur; qualitative examination (no differential count)), brain (medulla, pons, cerebrum and cerebellum), epididymides, esophagus, eyes, Harderian gland, heart, kidneys, lacrimal glands, large intestine (cecum, colon, rectum; cecum and colon were examined microscopically; however, the rectum was not examined microscopically), liver, lungs (with mainstem bronchi), lymph nodes (mesenteric, mediastinal), mammary gland (inguinal), nerve (sciatic), ovaries, pancreas, pituitary gland, prostate gland, salivary glands (submandibular), seminal vesicles, skeletal muscle (rectus femoris), skin (doral – base of tail), small intestine (duodenum, ileum, jejunum, Peyer’s patches/GALT), spinal cord (cervical, mid-thoracic, lumbar), spleen, stomach, testes, thymus, thyroid/parathyroid glands, trachea, urinary bladder, uterus (body/horns) with cervix, vagina and tissues with macroscopic findings including tissue masses
- In addition, slides of the indicated tissues were prepared and examined microscopically for all animals sacrificed at termination of the treatment period as well as the animal which died an unscheduled death. Any abnormalities not noted during macroscopic examinations which were seen during histology processing were recorded. In addition, the adrenal glands from males and the kidneys from females in Groups 2 and 3, sacrificed at termination of the treatment period and from animals in Groups 1 and 4 sacrificed at the end of the recovery period were examined microscopically.

LIVER AND KIDNEY TISSUE CONCENTRATIONS:
- Liver (left lobe) and kidney (a longitudinal section of the left kidney) samples (at least 0.5 grams) were collected and were analysed for molybdenum, copper, zinc and manganese under non-GLP conditions using a validated inductively coupled plasma mass spectrometry (ICP-MS) method.
- Tissues were dried in a 75°C oven in preparation for the acid digest and analysis.
Other examinations:
VAGINAL CYTOLOGY/ESTROUS CYCLING
- Daily vaginal smears were taken from each female at approximately the same time each day and the stage of oestrous determined, commencing after completing 6 weeks of dosing for 3 weeks (Weeks 7-9).
- At the end of the study, the overall pattern of each female was characterized as regularly cycling (having recurring 4 to 5 day cycles), irregularly cycling (having cycles with a period of diestrus longer than 3 days or a period of cornification longer than 2 days), or not cycling (having prolonged periods of either vaginal cornification or leukocytic smears).
- An animal was considered to be "not cycling" if she showed three or more consecutive days of oestrus or five or more consecutive days of dioestrus.
- Cycle length may be defined as the number of days from one oestrus to the next oestrus. Incomplete cycles are not counted in calculating mean cycle length. Mean cycle length for each animal is calculated first, and the mean of these means is then calculated to represent the group.

SPERM AASSESSMENT
Sperm evaluations were performed as outlined in OECD 416 (adopted 22 Jan 2001).
- Sperm counts: the right testis and cauda epididymis of all surviving animals at the terminal sacrifice and at the recovery sacrifice were removed intact, weighed fresh, and then frozen at approximately –80ºC (± 10°C) until evaluation for sperm count (spermatids in the testis).
- All surviving males (all groups at terminal sacrifice and at the recovery sacrifice) were processed for sperm counts. For each of these animals, homogenized samples of the caudal epididymis and the testis were stained and examined. For each stained preparation, 10 fields were counted. The total number of sperm in the caudal epididymis, or spermatids in the testis, was calculated and reported adjusted for organ weight.
- Sperm morphology: sperm morphology slides were prepared for each of the surviving males (all groups at terminal sacrifice and at the recovery sacrifice). The slides of all males at the terminal sacrifice and at the recovery sacrifice were evaluated for morphological development (approximately 200 sperm per animal within the 2 slides were assessed).
- Sperm motility: from all males (all groups at the terminal sacrifice and at the recovery sacrifice), the right vas deferens were excised. After a “swim-out” period, a sample was placed in an analyser and at least 200 sperm and/or five microscope field images were stored electronically.
- The stored fields belonging to the all males chosen for sperm counts were reported for percent motility.
Statistics:
The following parameters were analysed statistically: body weight, body weight change from interval to interval, cumulative body weight change from baseline, food consumption, food conversion efficiency, haematology, coagulation, clinical chemistry, organ weights, estrous cycles and sperm evaluations
The parameters to analyze were identified as continuous, discrete or binary. Test-substance treated groups were then compared to the control using the following procedures.
For all parameters, significant differences between control and test substance-treated groups were expressed at the 5% (p<0.05), 1% (p<0.01) or the 0.1% (p<0.001) level.
- Continuous parameters: Bartlett's test for variance homogeneity, Williams' test, Dunnett's test, Shirley's test for a monotonic trend, Steel's test, F1 test, H1 test, t-tests and Wilcoxon rank sum tests
- Discrete parameters: Jonckheere-Terpstra test, Kruskal-Wallis test, and exact Wilcoxon rank sum tests
- Binary parameters: Cochran-Armitage test, א2 test and Fisher's Exact tests
The following major computer/software systems were utilized at the laboratory: ClinAxys II, Hamilton – Thorne Sperm analyzer, Liberate Reporting System, Microsoft Word and/or Excel, Pristima System, Quasar, REES Scientific Environmental Monitoring System and Xybion Path/Tox System

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no test substance-related unscheduled decedents.
There were no test substance-related clinical signs.

BODY WEIGHT AND WEIGHT GAIN
Dosing phase:
Males:
- 60 mg Mo/kg bw/day: statistically significant decreases in body weight gains were observed almost weekly from Week 1 through Week 13 as measured from the pretest baseline and as measured from interval-to-interval. By the end of the dosing phase, absolute body weight were 15.1% less than controls.
- 5 and 17 mg Mo/kg bw/day: no test substance-related effects on the body weights

Females:
- 60 mg Mo/kg bw/day: statistically significant decreases in body weight gains weekly starting at Week 6 as measured from the pretest baseline. These differences were only occasionally seen in the interval-to-interval measures. By the end of the dosing phase, absolute body weight in the 60 mg Mo/kg bw/day females was 5.6% lower than controls (the value was not statistically significant).
- 5 and 17 mg Mo/kg bw/day: no test substance-related effects on the body weights

Recovery phase:
Males:
- 60 mg Mo/kg bw/day: statistically significant increases in body weight gains were noted at each weekly intervals but the absolute body weight was still 9.5% less than controls by the end of the study.
Females:
- 60 mg Mo/kg bw/day: weekly increases in body weight gains but only a few values were statistically significant. The absolute body weights in these females were considered to have recovered by the end of the study.

FOOD CONSUMPTION:
Dosing phase:
- 60 mg Mo/kg bw/day males had statistically significant decreases in food consumption on numerous occasions throughout the dosing phase.
- The weekly food consumption in test substance-treated females was generally considered to be comparable to control values.

Recovery phase:
- weekly food consumption in the 60 mg/kg bw/day males and females was considered to be comparable to their concurrent controls.

TEST SUBSTANCE INTAKE
- Test substance intake was on average close to the intended values with the males consistently less than intended and the females consistently greater than intended as a result of using averaged body weight and food consumption data for the calculations of dose concentration during the study.
- The averaged results for the study are summarized in Table 3 (please refer to "Any other information on results incl. tables" below.)

FOOD CONVERSION EFFICIENCY
Dosing phase:
- Food conversion efficiency showed that the 60 mg Mo/kg bw/day males and females generally had lesser values than the concurrent control animals during the dosing phase. This suggests that the reduced bodyweight gain was not only due to reduced food intake as a possible consequence of a palatability problem, but may suggest some interference with nutrition.
- No such effects were observed in the mid and low dose groups of animals.

Recovery phase:
- Food conversion efficiency showed that the 60 mg Mo/kg bw/day males and females generally had greater values than the concurrent control animals during the initial intervals indicating a recovery from the dosing phase.

OPHTHALMOSCOPIC EXAMINATION
There were no test substance-related ocular abnormalities.

HAEMATOLOGY
No test substance-related haematologic findings.
No test substance-related findings for coagulation.

CLINICAL CHEMISTRY
No test article-related clinical chemistry changes.

ORGAN WEIGHTS
Dosing phase and recovery phase:
No test substance-related findings.

GROSS PATHOLOGY
No test substance-related macroscopic findings.

HISTOPATHOLOGY: NON-NEOPLASTIC
Dosing phase:
- Microscopic findings considered to be related to test-substance administration were present in the kidneys of females administered 60 mg Mo/kg bw/day. Two females from the 60 mg Mo/kg bw/day dose group showed slight diffuse hyperplasia of the proximal tubules in the kidney. Although the finding was only present in two test substance treated rats, it is uncommon as a background finding in this age of animal and is therefore considered test-substance related. It is possible that the elevated concentrations of copper in the kidneys may play some role in the histopathological changes in the kidneys among the high dose females.

Recovery phase:
- The finding of proximal tubule hyperplasia in the kidneys of females administered 60 mg Mo/kg bw/day was not observed in any of the animals following a 60 day recovery period.

BLOOD MOLYBDENUM:
Mean serum and whole blood molybdenum results from the Weeks 4 and 12 during dosing and the Days 2 and 7 during recovery can be seen In Table 1 and Table 2 (please refer to "Any other information on results incl. tables" below.)
The results showed:
- Males had higher serum and/or whole-blood exposure to Mo than the females (~23% on average for Groups 2-4) at all dose levels at both weeks 4 and 12 of dosing. This is the opposite of expected based on the test substance intake results where the females had higher Mo intake.
- No or only slight accumulation of Mo in the serum (week 12 serum results were only slightly greater (~14% on average for Groups 2-4 both sexes) than week 4 serum results).
- Whole blood results in week 12 were consistently lower (~55% on average for Groups 2-4 both sexes) than the serum results in Week 12.
- A rapid recovery at days 2 and 7 after completion of dosing as expressed by substantially and progressively lower serum results at both intervals of measurement in the Group 4 animals.
- Serum copper levels were increased in the high dose Group 4 animals compared with the control Group 1 animals. The mean copper levels at 4 weeks in the control males was 1.266 μg/mL and in the females was 1.767 μg/mL. In the Group 4 males the level was 4.512 μg/mL and in females was 4.513 μg/mL . At 12 weeks, the levels were still high with serum copper levels in Group 4 males of 5.786 μg/mL and in females of 6.627 μg/mL.

VAGINAL CYTOLOGY7ESTROUS CYCLE.
No test substance-related effects on vaginal cytology and oestrous cycles during weeks 7-9 of the dosing phase.

SPERM EVALUATIONS:
No effect of treatment was observed on testes or secondary sex organ weights, and no effects on spermatid or sperm counts, motility or morphology were observed.

ORGAN ANALYSIS - LIVER AND KIDNEY:
Mean organ molybdenum concentrations (dry weight basis) from the terminal and recovery sacrifice intervals are shown in Table 4 and Table 5 (please refer to "Any other information on results incl. tables" below).

The results showed:
- Group 2 liver concentrations at termination were only slightly higher than Group 1 liver concentrations suggesting close to background levels at the low dose level.
- Groups 3 and 4 liver concentrations at termination were elevated above Groups 1-2 concentrations, but not in a fully dose proportional manner.
- Group 4 liver concentrations at end of recovery were substantially lower than at termination suggesting a nearly complete recovery (especially in the males) towards background levels.
- Groups 2-4 kidney concentrations at termination were elevated above Group 1 concentrations and in a nearly dose proportional manner.
- Group 4 kidney concentrations at end of recovery were substantially lower than at termination suggesting incomplete recovery towards background levels.

In addition the following results were found:
- Liver and kidney copper levels were increased in the high dose Group 4 animals compared with the control Group 1 animals.
- Mean liver copper level in Group 1 males was 16.97 μg/g and in females was 19.22 μg/g. In Group 4 males it was 25.06 μg/g, and in females was 36.33 μg/g.
- Kidney copper levels in Group 1 males was 30.191 μg/g and in females was 43.538 μg/g. In Group 4 males it was 81.70 μg/g and in the females was 138.72 μg/g.
- In the recovery Group 4 animals, copper levels in liver and kidney were reduced but still higher than in the Group 1 controls. This may be important since some of the kidney toxicity may be related to the high copper levels in the tissues.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
17 mg/kg bw/day (nominal)
Based on:
element
Sex:
male/female
Basis for effect level:
other: NOAEL is based on the effects on body weights and kidneys seen at 60 mg Mo/kg bw/day.
Dose descriptor:
NOAEL
Effect level:
> 60 mg/kg bw/day (nominal)
Based on:
element
Sex:
male/female
Basis for effect level:
other: NOAEL based on no effects on testicular (or gonadal) and sperm and oestrous cycle effects at the highest dose tested (60 mg/kg bw/d).

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Serum and Whole Blood Molybdenum Results - males

GROUP

Serum

Week 4

Serum

Week 12

Whole

Blood

Week 12

Serum

Day 2

Recovery

Serum

Day 7

Recovery

 

(ng/mL)

(ng/mL)

(ng/mL)

(ng/g)

(ng/g)

1

18.7

19.4

11.9

19.0

20.6

2

1332

1309

912

NA

NA

3

4687

4674

2930

NA

NA

4

16277

18497

9903

4382

2425

NA = not applicable

Table 2: Serum and Whole Blood Molybdenum Results - females

GROUP

Serum

Week 4

Serum

Week 12

Whole

Blood

Week 12

Serum

Day 2

Recovery

Serum

Day 7

Recovery

 

(ng/mL)

(ng/mL)

(ng/mL)

(ng/g)

(ng/g)

1

19.8

17.9

11.1

15.1

33.3

2

991

1121

720

NA

NA

3

3370

4311

2628

NA

NA

4

13176

15531

7736

6447

2841

NA = not applicable

Table 3: Test Substance Intake Results – mg Mo/kg bw/day

Group – target dose

Males

Females

Mean

2 – 5 mg Mo/kg bw/day

4.5

5.4

5.0

3 – 17 mg Mo/kg bw/day

15.1

19.0

17.1

4 – 60 mg Mo/kg bw/day

54.8

65.2

60.0

Table 4: Molybdenum in Organs Results - Terminal

GROUP

Liver

Conc - males

Kidneys

Conc. - males

Liver

Conc - females

Kidneys

Conc - females

 

(ug/g)

(ug/g)

(ug/g)

(ug/g)

1

2.22

0.93

2.45

0.94

2

2.54

2.30

3.41

3.83

3

4.00

9.52

4.92

10.94

4

12.02

43.18

12.99

55.04

Table 5: Molybdenum in Organs Results - Recovery

GROUP

Liver

Conc - males

Kidneys

Conc. - males

Liver

Conc - females

Kidneys

Conc - females

 

(ug/g)

(ug/g)

(ug/g)

(ug/g)

1

1.88

0.91

2.71

0.97

4

2.30

7.04

4.58

16.86

Applicant's summary and conclusion

Conclusions:
The dietary administration of 5, 17 or 60 mg/kg bw/day of Mo (molybdenum in sodium molybdate dihydrate) to rats for at least 90 days resulted in reduced bodyweight gain in the 60 mg Mo/kg bw/day animals. The effect was more severe in males. In males, this may have been due in part to slightly reduced food intake. Light microscopy evaluation of control and 60 mg Mo/kg bw/day animals identified test substance-related findings in the kidneys (slight diffuse hyperplasia of the proximal tubules) of two 60 mg Mo/kg bw/day females which recovered following up to 60 days after completion of dosing. No adverse effects were observed on the gonads, oestrous cycles or sperm analyses in any of the treated animals.
A NOAEL was determined to be 17 mg Mo/kg bw/day based on the effects on body weights and kidneys seen at 60 mg Mo/kg bw/day.
The NOAEL for testicular (or gonadal) and sperm and oestrous cycle effects is > 60mg Mo/kg bw/day.