Molybdic acid

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Molybdic acid is not expected to show adverse effects on sexual function and fertility. This is supported by the absence of any adverse effects on parameters relating to fertility at up to 60 mg Mo/kgbw/day in a 90-day repeated dose toxicity study (Hoffman, 2011) which was modified to include several additional parameters related to reproductive toxicity.

Link to relevant study records

Reference

Endpoint:

reproductive toxicity, other

Remarks:

Repeated dose toxicity study (according to OECD 408 under GLP) modified to include parameters related to reproductive toxicity, such as oestrous cycle and sperm analyses as specifed in OECD 416.

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010-10-07 to 2011-10-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remarks'

Remarks:

The study study is not a full reproductive toxicity study, but a repeated dose toxicity study (acc. OECD 408, under GLP), modified to include parameters related to reproductive to xicity, such as oestrous cycle and sperm analyses as specified in OECD 416.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD 408 - repeated dose toxicity study, modified to include parameters related to reproductive toxicity, such as oestrous cycle and sperm analyses as specified in OECD 416.

Principles of method if other than guideline:

In addition to toxicological parameters studied in an OECD 408 repeated dose toxicity study, this st
udy was amended to also include parameters relevant to assess potential effects on reproduction: oe
strous cycles, sperm parameters (count, motility and morphology, spermatid counts).

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

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

Route of administration:

oral: feed

Vehicle:

other: Certified Rodent diet, N°. 2016C

Details on exposure:

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.

Details on mating procedure:

No mating took place. OECD 408 repeated dose toxicity study was amended to include parameters relevant to assess potential effects on reproduction.

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) and 92 (females) days

Frequency of treatment:

Tes and controls groups: ad libitum feed presentation

Remarks:

Doses / Concentrations:
0, 5, 17 and 60 mg/Kg bw/day
Basis:
actual ingested
based on elemental Mo

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.

Parental animals: Observations and examinations:

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.

Oestrous cyclicity (parental animals):

- 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 parameters (parental animals):

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.

Postmortem examinations (parental animals):

GROSS PATHOLOGY:
- 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.

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

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

effects observed, treatment-related

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

not specified

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.

Dose descriptor:

NOAEL

Effect level:

> 60 mg/kg bw/day

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).

Remarks on result:

other: Generation not specified (migrated information)

Dose descriptor:

NOAEL

Effect level:

17 mg/kg bw/day

Based on:

element

Sex:

male/female

Basis for effect level:

other: NOAEL based on the effects on body weights and kidneys seen at 60 mg Mo/Kg bw/day.

Clinical signs:

not specified

Mortality / viability:

not specified

Body weight and weight changes:

not specified

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Dose descriptor:

other:

Remarks:

Brief summary of findings

Remarks on result:

not measured/tested

Remarks:

Effect level not specified OECD 408 study modified to include parameters related to reproductive toxicity such as oestrous cycle and sperm anaylses as specified in OECD 416.

Reproductive effects observed:

not specified

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Additional information

The UV spectral analysis of the samples taken during water solubility testing of Molybdic acid shows that molybdate ions are formed upon dissolution of Molybic acid. Under physiological conditions the molybdate anion will be the relevant molybdenum species liberated for Molybdic acid. Read-across from sodium molybdate is therefore justified.

A guideline-conform repeated dose toxicity study (acc. OECD 408, under GLP) in rats with the test item sodium molybdate was modified to include parameters related to reproductive toxicity, such as oestrous cycle and sperm analyses as specified in OECD 416. In that 90-day repeated dose toxicity study (Hoffman, 2011) disodium molybdate was administered to male and female rats at doses of 5, 17 or 60 mg/kg bw/day of Mo (molybdenum in disodium molybdate dihydrate) via feed. In addition to the standard examination parameters, the following examinations were conducted to assess any adverse effects on sexual function and fertility: vaginal cytology, oestrus cycle, qualitative sperm staging (in acc. with OECD 416).

Reduced bodyweight gains were observed in the 60 mg Mo/kg bw/day dose group. The effect was more pronounced in males, which might be due to a slightly reduced food intake. During the recovery phase food consumption in the 60 mg/kg bw/day males and females returned to a value comparable to the control animals. Light microscopic evaluation of control and 60 mg Mo/kg bw/day animals showed test item-related findings in the kidneys (slight diffuse hyperplasia of the proximal tubules) of two 60 mg Mo/kg bw/day females. No such findings were reported for the animals after the 60-day recovery phase.

There were no test substance related changes in the male or female reproductive tissues (testes, epididymis, prostate, seminal vesicles, ovaries, uterus or vagina). There were no test substance-related effects on vaginal cytology and oestrous cycles during weeks 7-9 of the dosing phase (i. e., the period during which vaginal cytology and oestrous cycles were evaluated). No test-item related changes in organ weight of testes or secondary sex organs and no effect on spermatid or sperm counts, motility or morphology were observed. All other recorded microscopic findings were considered incidental and unrelated to administration of disodium molybdate dihydrate. They occurred at similar incidences in the control and test substance treated groups or they were sporadic with no relationship to dose.

In this 90-day repeated dose toxicity study the NOAEL for systemic toxicity 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 effects on reproductive organs, sperm and oestrous cycle is 60 mg Mo/kg bw/day.

Effects on developmental toxicity

Description of key information

Molybdic acid is not expected to be a developmental toxicant. This is supported by a guideline-conform prenatal developmental toxicity study (acc. OECD 414, under GLP) in rats in which the NOAEL for developmental toxicity has been determined at ca. 40 mg Mo/kg bw/day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted under GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 2001-01-22

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl: CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC
- Age at study initiation: ~ 8 to 10 weeks upon arrival on gestational day 0 or 1
- Weight at study initiation: ~ 200-250 g on gestational day 3 (day of randomization into groups)
- Housing: solid-bottom, polycarbonate caging; acclimation and gestation: singly; enrichment: nestlets; bedding: Sani-Chips® cage litter (P.J. Murphy Forest Products, Montville, NJ)
- Diet (ad libitum): Purina Certified Rodent Chow No. 5002 (Purina Mills, Inc., Richmond, IN)
- Water (ad libitum): tap water
- Acclimation period: at least 3 days

All animals were used in compliance with the NRC Guide for the Care and Use of Laboratory Animals (2011).

ENVIRONMENTAL CONDITIONS
- Temperature: 21.7 - 22.8 °C
- Relative humidity: 35.82 to 55.44
- Air changes: 10-15 per hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
A premix was prepared by mixing chemical over a small portion of blank feed. The formulation was prepared by layering additional blank feed, the premix, and more blank feed in a twin-shell V-blender and mixing for approximately 15 minutes.
One dietary formulation for each group was prepared once.
The formulations were stored at ambient temperature (20°C). The dosed feed in the jars was changed at least once per week during conduct of the study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of the test chemical in the dosed feed formulations were conducted. Homogeneity of the dosed feed formulations was evaluated using the same batch size as required for the animal study and at the lowest and highest proposed dietary concentrations. Samples for analysis were collected from left, right, and bottom blender ports for each formulation. A sample of the blank feed was also sent for analysis.
The frequency of analysis was as follows:
- dose concentration analysis for all formulations (once), prior to study start.
- homogeneity once on the dose formulations for low and high dietary concentrations, prior to study start.
- stability once on the high and low dietary dose formulations out to 28 days, begun prior to study start (so the diets are never used beyond their confirmed stability).

Results:
The analyses indicated that the high and low dose formulations were stable for at least 28 days at room temperature. Analyses of aliquots of all dosing formulations also determined that the dietary dose formulations of sodium molybdenum dihydrate in the feed at 0, 100, 338, 675 and 1350 ppm (parts per million) were accurate; all dietary samples were all well within ± 10% of the target concentrations of molybdenum, and homogenous. The concentration of sodiummolybdate dihydrate added to the diet of 0, 100, 338, 675 and 1350 ppm are equivalent to molybdenum concentrations of 0, 40, 134, 268 and 536 ppm. The analyses of molybdenum by Michigan State University of the feed samples, gave values of molybdenum of 1.75 to 1.80, 39.7-40.8, 134-139, 264-268, and 540-542 ppm for the 0, 40, 134, 268 and 536 ppm feeds respectively The dose formulations were therefore appropriate for use and were used in this study.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant (mated at the vendor over 4 days)
- Proof of pregnancy: a positive vaginal smear was designated gestational day (GD) 0.

Duration of treatment / exposure:

Gestational days 6 through 20

Frequency of treatment:

ad libitum (7 days/week)

Duration of test:

20 days

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

equivalent to 0 mg Mo/kg bw/day

Dose / conc.:

7.5 mg/kg bw/day (nominal)

Remarks:

equivalent to 3 mg Mo/kg bw/day

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

equivalent to 10 mg Mo/kg bw/day

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

equivalent to 20 mg Mo/kg bw/day

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

equivalent to 40 mg Mo/kg bw/day

No. of animals per sex per dose:

25 timed-mated (presumed pregnant) females

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: in a dose-range finding study conducted previously, administration of sodium molybdate dihydrate in the diet, available ad libitum to pregnant rats from gestational day 6 to term necropsy on gestational day 20 was not associated with any treatment- or dose-related maternal findings at any molybdenum dose (0, 37.5, 187, 375 and 750 ppm of sodium molybdenum dihydrate and equivalent to at 0, 1-20 mg/kg/day molybdenum) or any time during gestation or at scheduled necropsy, including no effects on maternal body weights, weight gains, feed consumption in g/day or g/kg/day, clinical observations, pregnancy indices or organ weights. There were also no treatment- or dose-related developmental toxicity findings at any dose, including no effects on pre- or postimplantation loss, foetal numbers, sex ratio, body weights, or foetal external malformations or variations. There were no foetal external malformations or variations found in this study; this is comparable to the laboratories historical control database which indicates 17 foetuses with external malformations/variations out of 14,352 foetuses, approximately 0.12%.
Therefore, dietary concentrations of 0, 100, 338, 675, and 1350 ppm (equal to 0, 3, 10, 20 and 40 mg/kg bw/day of Mo) were chosen for the definitive developmental toxicity study.
- Rationale for animal assignment: timed-mated females were assigned to treatment groups in Provantis™ by stratified randomization, by body weight on gestational day 3, to provide uniform mean body weights across dose groups (± 20g).

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily, twice per day, at least 6 hours apart
- Cage side observations: morbidity/mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical observations (out of cage) were conducted and recorded at least once daily.

BODY WEIGHT: Yes
- Time schedule for examinations: gestational days 3, 6, 9, 12, 15, 18 and 20

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption was measured on gestational days 3, 6, 9, 12, 15, 18 and 20.

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20 (euthanasia by CO2 asphyxiation)
Identification and retention of any maternal gross lesions in 10% formalin.

ORGAN WEIGHTS: Yes
- Organs: uterus, liver, kidneys

ANALYTICAL CHEMISTRY:
- Serum from 10 arbitrarily selected females per group collected via cardiac puncture.
- Maternal livers and kidneys were arbitrarily chosen from 2 or 3 females per dose group on each mating day to equal 10 pregnant females per group. Foetal placentae grouped by litter were collected from these same 10 females/group

HISTOPATHOLOGY: Yes
The remaining maternal livers and kidneys/group were fixed in 10% neutral buffered formalin for subsequent histopathology of the 15 remaining pregnant females at 0 ppm and of the 15 remaining pregnant females at 1350 ppm.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of full resorptions: Yes
- Number of uterine implantation sites (live and dead): Yes

Fetal examinations:

Live fetuses were dissected from the uterus after maternal termination on gestational day 20 and euthanised.
- Foetal weight and sex: all live foetuses
- External examinations: Yes, 100% live foetuses
- Soft tissue examinations: Yes, 50% live foetuses (decapitated)
- Skeletal examinations: Yes, 50% (intact fetuses)
- Head examinations: Yes, 50% (same fetuses which received visceral examination)

Statistics:

All statistical analyses were performed using Provantis™ software. For all statistical tests, p<0.05 was used as the criterion for significance.
Quantitative continuous data (e.g., maternal body weights and weight gains, feed consumption in g/kg and g/kg body weight/day) were subjected to the Provantis™ generalized ANOVA/ANCOVA test. This decision tree includes analysis of variance (ANOVA) and covariance, nonparametric analysis of variance, pairwise tests (Dunnett, 1955; 1964) for parametric and nonparametric data, and Levene’s test (Levene, 1960) for homogeneity of variance. For each variable analyzed, where there was evidence of difference between groups, the methodology also identified those groups which differ from the control group.
The uterine weight and uterine implant data (i.e., data presented in the Uterine Implantation Data Table) were subjected to the Kruskal-Wallis nonparametric analysis of variance, which is the default technique used by Provantis™ (Kruskal and Wallis, 1952; Conover, 1971). When there was evidence of a significant group effect, pairwise comparisons of each treated group with the control group were performed using Dunnett’s test on the ranks.
The foetal weight and sex ratio (i.e., data presented in the Group Mean Caesarean-Fetal Data Table) were subjected to a 1-way mixed ANOVA, which is the default technique used by Provantis™. When there was evidence of a significant group effect, pairwise comparisons of each treated group with the control group were performed using Dunnett’s test on group least square means. No statistical tests were performed on incidences of foetal malformations or variations.

Historical control data:

no data

Details on maternal toxic effects:

Maternal toxic effects :no effects. Remark: Although no clear toxic effects were observed in either the dams or on the embryos/fetuses, the top dose is equivalent to approximately 20,000 times the average human daily intake of molybdenum from food and water of about 2 µg/kg bw/day.

Details on maternal toxic effects:
There were no treatment or dose-related effects on maternal body weights, weight changes, feed consumption in grams/day or grams/kg, body weight/day, or on maternal clinical observations, pregnancy indices, or maternal organ weights at any dose. There were also no biological or statistical differences among groups for the numbers of ovarian corpora lutea/female, for uterine implantation sites, or for uterine implantation losses per female at any dose.
In addition, there were no potential test article-related histopathological lesions identified during the pathology examination (kidney and liver).

Dose descriptor:

NOAEL

Effect level:

> 100 mg/kg bw/day

Based on:

test mat.

Remarks:

equivalent to 40 mg Mo/kg bw/day

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects: no effects

Details on embryotoxic / teratogenic effects:
There were no biological or statistical differences among groups for the numbers of foetuses, foetal sex ratios, foetal body weights, foetal external, visceral or skeletal malformations or variations per female at any dose. The incidences of the few foetal malformations and the more common foetal variations observed in the study were comparable to the historical control database of the laboratory on this rat strain and supplier. The foetal effects in this study also did not exhibit any treatment- or dose- related pattern of increased incidences and/or severities.

Key result

Dose descriptor:

NOAEL

Effect level:

> 100 mg/kg bw/day

Based on:

test mat.

Remarks:

equivalent to 40 mg Mo/kg bw/day

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Analysis of the elements in blood and tissues showed that there were significant and dose related increases in molybdenum and copper in blood and all tissues examined.

Conclusions:

NOAEL > 40 mg/kg bw/day molybdenum (maternal toxicity).
NOAEL > 40 mg/kg bw/day molybdenum (developmental toxicity).
Based on the results of this main developmental toxicity study (and the previous dose-range finding study) of dietary molybdenum, an essential element, it has no adverse effect on maternal or embryofoetal endpoints in rats up to and including 1350 ppm in the feed, corresponding to approximately 40 mg Mo/kg/day. Although no clear toxic effects were observed in either the dams or on the embryos/fetuses, the top dose is equivalent to approximately 20,000 times the average daily intake of molybdenum from food and water of about 2 µg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

40 mg/kg bw/day

Species:

rat

Additional information

The UV spectral analysis of the samples taken during water solubility testing of Molybdic acid shows that molybdate ions are formed upon dissolution of Molybic acid. Under physiological conditions the molybdate anion will be the relevant molybdenum species liberated for Molybdic acid. Read-across from sodium molybdate is therefore justified.

A guideline-conform prenatal developmental toxicity study (acc. OECD 414, under GLP) in rats with the test item sodium molybdate is available. Exposure was during gestational days 6 through 20 via the diet, in four dose groups (ca. 3, 10, 20 and 40 mg Mo/kg bw/day) and a control group (plain diet). For complete study details, please refer to the endpoint study record in the technical dossier.

There were no treatment or dose-related effects on maternal body weights, weight changes, feed consumption in grams/day or grams/kg, body weight/day, or on maternal clinical observations, pregnancy indices, or maternal organ weights at any dose. There were also no biological or statistical differences among groups for the numbers of ovarian corpora lutea/female, for uterine implantation sites, or for uterine implantation losses per female at any dose. Therefore, the NOAEL for maternal toxicity was established at 40 mg Mo/kg bw/day.

There were no biological or statistical differences among groups for the numbers of foetuses, foetal sex ratios, foetal body weights, foetal external, visceral or skeletal malformations or variations per female at any dose. The incidences of the few foetal malformations and the more common foetal variations observed in the study were comparable to the historical control database of the laboratory on this rat strain and supplier. The foetal effects in this study also did not exhibit any treatment- or dose- related pattern of increased incidences and/or severities. The NOAEL for developmental toxicity is 40 mg Mo/kg bw/day. Although no clear toxic effects were observed in either the dams or the embryos/fetuses, the highest dose tested is equivalent to approximately 20,000 times the average daily general population intake of molybdenum from food and water of about 2 µg/kg bw/day.

Justification for selection of Effect on developmental toxicity: via oral route:
Key study = Tyl (2013) - NOAEL = 40 mg Mo/Kg bw/day

Justification for classification or non-classification

The information available regarding the effects of molybdenum on fertility and development does not result in the need to classify Molybdic acid for reproductive toxicity.