Dimolybdenum carbide

Endpoint summary

Description of key information

A GLP guideline study according to OECD 408 was conducted. Based on effects on body weight, body weight gain, food conversion efficiency and renal histopathology (females only) at 60 mg Mo/kg bw/day the NOAEL for both sexes is considered to be 17 mg Mo/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented study conducted according to OECD Guideline 408 and OECD guideline 416 in compliance with GLP. No deviations from the guidelines were reported.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Principles of method if other than guideline:

In addition, in view of the observations on testes and estrus cycles in the published literature, it was decided to include examination of estrus cycles, analysis of various sperm parameters and detailed histopathology of reproductive organs as described in the OECD Guideline 416 for a two-generation reproduction toxicity study.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)IGS BR, VAF/Plus Sprague–Dawley derived

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Kingston, NY, USA
- Age at study initiation: approximately 7 weeks of age when received and beginning of acclimation period, approx. 9 weeks of age at start of dosing
- Weight at study initiation: Males: 338.4 g (range 309.8–377.6 g); Females: 229.6 g (range 187.9–263.5 g)
- Fasting period before study: No
- Housing: Individually housed in elevated, stainless steel, wire mesh cages
- Diet (e.g. ad libitum): Certified Rodent Diet (No. 2016C; Harlan Teklad, Madison, WI), ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 2–2.5 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19–23 °C
- Humidity (%): 34–49%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Dietary dose formulations were prepared once weekly for the first 4 weeks of the study, then every other week for the rest of the treatment period to achieve the target dose levels in mg Mo/kg bw/day. Dietary dose formulations were stored at room temperatureunder low humidity conditions when not in use. Homogeneity of the dietary dose formulations was confirmed, and stability was confirmed to be at least 5 weeks.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analysis confirmed that the diets were of appropriate concentration of molybdenum and that homogeneous dietary mixtures were administered. Mean analytical concentrations of molybdenum in the diets were 91–102% of the nominal concentrations. Exposure to the test substance was generally comparable to the targeted dose levels, but with the males it was consistently less than intended and with the females consistently more than intended. Specifically, the actual mean intake of molybdenum at the nominal dose levels of 5, 17, and 60 mg Mo/kg bw/day was 4.5, 15.1, and 54.8 mg Mo/kg bw/day, respectively, among males and 5.4, 19.0, and 65.2 mg Mo/kg bw/day, respectively, among females, giving overall mean intakes of 5.0, 17.1 and 60.0 mg Mo/kg bw/day.

Duration of treatment / exposure:

91 days (males) and 92 days (females)

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0 mg Mo/kg bw/day (Group 1)
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
5 mg Mo/kg bw/day (Group 2)
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
17 mg Mo/kg bw/day (Group 3)
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
60 mg Mo/kg bw/day (Group 4)
Basis:
nominal in diet

No. of animals per sex per dose:

Groups 1 (control): 20
Groups 2 (low dose): 10
Groups 3 (middle dose): 10
Group 4 (high dose): 20

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 28-day range finding study, conducted at the same laboratory, in which Sodium molybdate dihydrate was given to groups of 5 rats/sex/group in the diet or by gavage (once or twice daily) for 28 consecutive days at dose levels of 0, 4, or 20 mg Mo/kg bw/day. There was no mortality or treatment-related clinical signs in any of the groups of rats in the range-finder study. Decreases in body weight (8% decrease on day 28; not statistically significant; n = 5) and body weight gain (19% decrease on day 28; p < 0.05 at most time intervals; n = 5) were observed among males given the high dose (20 mg Mo/kgbw/day) in the diet. In females, no effect on body weight or body weight gain was observed at any dose level. There were no convincing treatment-related effects on organ weights or macroscopic or microscopic findings. Since little evidence of toxicity was found in the range finder study, it was decided to use a higher dose range in the 90day study.
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: 10 animals/sex/group in Groups 1 and 4 were continued on untreated diet for up to 60 days. At the end of the recovery period, they were necropsied.
- Post-exposure recovery period in satellite groups: 60 days

Positive control:

N.A.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations included: Mortality and general condition.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 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 behavior.

BODY WEIGHT: Yes
- Time schedule for examinations:
Weighed twice pretest, weekly during the study and terminally (after fasting) just prior to necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/wk:
Yes. Food consumption was measured pretest, at Days 2, 4, and 7 during the first week of treatment, and weekly (at 6 day intervals) thereafter. The amount of food consumed over a 6-day period during the week was used to determine feed concentration calculations for the following week for the first 4 weeks and every other week during the rest of the study.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data:
No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pretest and at the termination of the treatment period
- Dose groups that were examined: All animals
- The cornea, anterior chamber, lens, iris, vitreous humor, retina, and optic disc were examined by indirect ophthalmoscopy.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Not reported
- Anaesthetic used for blood collection: Yes (Isofuorane)
- Animals fasted: Yes, overnight prior to blood collection.
- How many animals: Not reported
- Parameters checked included: hemoglobin concentration, hematocrit, erythrocyte count, platelet count, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red cell distribution width, total leukocyte count, reticulocyte count, and differential leukocyte count. Coagulation parameters (prothrombin time and activated partial thromboplastin time) were also determined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Not reported
- Animals fasted: Yes, overnight prior to blood collection.
- How many animals: Not reported
- Parameters checked included: aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, blood urea nitrogen, creatinine, glucose, cholesterol, triglycerides, total protein, albumin, uric acid, total bilirubin, sodium, potassium, chloride, calcium, and inorganic phosphorus.

URINALYSIS: No data
- Time schedule for collection of urine: N/A
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked included: N/A

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Twice daily
- Dose groups that were examined: All animals (during cage side observations)
- Battery of functions tested: Changes in the level of motor activity, gait and posture, reactivity to handling or sensory stimuli, grip strength, and stereotypies or bizarre behavior.

OTHER:
VAGINAL CYTOLOGY/ESTRUS CYCLES: Daily vaginal smears were taken from each female at approximately the same time each day and stage of estrus determined for 3 weeks starting after completing 6 weeks of treatment. The overall pattern of each female was characterized as regularly cycling (having recurring 4–5 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). Cycle length was defined as the number of days from one estrus to the next. Incomplete cycles were not included in calculating the mean cycle length.

SPERM EVALUATION: Sperm evaluations were performed as outlined in OECD TG 416. All surviving male animals were processed for sperm counts and sperm motility at the terminal sacrifice and the recovery sacrifice. For sperm and spermatid counts, homogenized samples of the right caudal epididymis and the right testis, respectively, were stained and examined; for each stained preparation, 10 fields were counted. For sperm motility, the right vas deferens of all surviving males was excised and placed in a pre-warmed solution of phosphate buffered saline and 7.5% bovine serum albumin fraction V in Medium 199. After a swim-out period, motility was evaluated using at least 200 sperm and/or five microscope field images. Two sperm morphology slides were prepared for all surviving males at terminal sacrifice and recovery sacrifice; approximately 200 sperm per animal within the 2 slides were evaluated.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. All animals were fasted overnight prior to necropsy and euthanized by carbon dioxide inhalation followed by exsanguination. All animals were subject to macroscopic examination, which included 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.

ORGAN WEIGHTS: Yes, absolute and relative organ weights (based on terminal body weights) were determined for the adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, pituitary gland, prostate gland and seminal vesicles, spleen, testes, thymus, thyroid/parathyroid glands, and uterus with cervix.

HISTOPATHOLOGY: Yes, all organs and tissues obtained during necropsy were preserved. Slides of these organs and tissues were prepared and examined microscopically for all Group 1 (control) and Group 4 (high-dose) animals sacrificed at termination of the treatment period. 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.

Other examinations:

ELEMENTAL ANALYSIS OF SERUM, WHOLE BLOOD, LIVER AND KIDNEY: Blood and serum samples were taken from 10 rats/sex/group and analyzed for molybdenum, copper, zinc, manganese, iron, cobalt and selenium at weeks 4 (serum only) and 12 (serum and blood), as well as at 2 and 7 days after cessation of treatment for the recovery groups (serum only). Liver and kidney samples from 10 rats/sex/group were collected at the end of the treatment period and the recovery period, and they were flash frozen in liquid nitrogen and shipped on dry ice to the analytical laboratory for analysis of the same elements as the serum samples. Serum, whole blood, and tissues were analyzed for molybdenum and the other elements using a validated inductively coupled plasma mass spectrometry method. The same laboratory and instrument was also used to analyze the feed samples.

Statistics:

Continuous data, including body weights, food consumption, hematology and clinical chemistry parameters, organ weights and sperm analysis, were subjected to a Bartlett’s test for variance homogeneity (Bartlett, 1937). If this was not significant at the 1% level, then parametric methods were applied using either Williams’ test (Williams, 1971, 1972) or Dunnett’s test (Dunnett, 1955, 1964). For non-continuous data, statistical tests were ranked and examined by the Kruskal–Wallis test (Kruskal and Wallis, 1952, 1953). If significant, the Wilcoxon rank sum test (Wilcoxon, 1945) was applied for comparison with the vehicle control group. For analyses of serum, whole blood and tissue levels of metals and other elements in the 90-day study, one-sided pairwise comparisons of each treatment group with their respective control were conducted using the R software package version 2.14.1 (R Development Core Team, 2011).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See details below

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See details below

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

See details below

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:

effects observed, treatment-related

Description (incidence and severity):

See details below

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See details below

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY: One male in the group given 60 mg Mo/kg bw/day was found dead on Day 47. There were no clinical signs prior to death and no macroscopic or microscopic findings to explain the cause of death. This single death was considered incidental and unrelated to the administration of the test substance. There was no mortality in any other groups. There were no treatment-related clinical signs or ocular abnormalities in any of the animals.

BODY WEIGHT AND WEIGHT GAIN: During the 90-day treatment phase, statistically significant decreases in body weight gain compared to controls were observed in males and females exposed to 60 mg Mo/kg bw/day beginning as early as weeks 1 and 6, respectively. At the end of the 90-day treatment period, the final body weights of males and females given 60 mg Mo/kg bw/day were 15.1% (p < 0.001) and 5.6% (not statistically significant) less, respectively, than their respective control values. During the 60-day recovery phase, the males in the group given 60 mg Mo/kg bw/day showed statistically significant increases in body weight gains at each weekly interval, but the absolute body weight was still significantly less than controls (by 9.5%; p < 0.05) at the end of the recovery phase. There were no treatment-related effects on body weight or body weight gain among the males or females given 5 or 17 mg Mo/kg bw/day.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Chemical analysis confirmed that the diets were of appropriate concentration of molybdenum and that homogeneous dietary mixtures were administered. Mean analytical concentrations of molybdenum in the diets were 91–102% of the nominal concentrations. Exposure to the test substance was generally comparable to the targeted dose levels, but with the males it was consistently less than intended and with the females consistently more than intended. Specifically, the actual mean intake of molybdenum at the nominal dose levels of 5, 17, and 60 mg Mo/kg bw/day was 4.5, 15.1, and 54.8 mg Mo/kg bw/day, respectively, among males and 5.4, 19.0, and 65.2 mg Mo/kg bw/day, respectively, among females, giving overall mean intakes of 5.0, 17.1 and 60.0 mg Mo/kg bw/day.

During the 90-day treatment phase, statistically significant decreases in body weight gain compared to controls were observed in males and females exposed to 60 mg Mo/kg bw/day beginning as early as weeks 1 and 6, respectively. At the end of the 90-day treatment period, the final body weights of males and females given 60 mg Mo/kg bw/day were 15.1% (p < 0.001) and 5.6% (not statistically significant) less, respectively, than their respective control values. During the 60-day recovery phase, the males in the group given 60 mg Mo/kg bw/day showed statistically significant increases in body weight gains at each weekly interval, but the absolute body weight was still significantly less than controls (by 9.5%; p < 0.05) at the end of the recovery phase. There were no treatment-related effects on body weight or body weight gain among the males or females given 5 or 17 mg Mo/kg bw/day. In males given 60 mg Mo/kg bw/day, food consumption was statistically significantly decreased on numerous occasions throughout the treatment period. Otherwise, compared to controls, no consistent difference in food consumption was observed in any of the other groups of males or females receiving the test material.

At 60 mg Mo/kg bw/day, food conversion efficiency was significantly reduced during the treatment period, particularly among the males, suggesting that some interference with nutrition, as well as reduced food consumption, may have played a role in reduced body weight gain.

OPHTHALMOSCOPIC EXAMINATION: There were no treatment-related ocular abnormalities in any of the animals.

HAEMATOLOGY: There were no effects on hematological parameters, apart from a marginal, statistically significantly shorter prothrombin times in males receiving 5, 17 or 60 mg Mo/kg bw/day. These changes were not considered to be related to the administration of the test substance because they were slight, not dose-related, and not observed in females. No significant difference from controls was seen for activated partial thromboplastin time in any group of males or females.

CLINICAL CHEMISTRY: clinical biochemistry evaluation revealed statistically significant decreases in total protein and calcium at 60 mg Mo/kg bw/day in males, but not in females. Creatinine and uric acid levels were statistically significantly reduced compared to controls among females at all dose levels, but a similar effect was not observed in males. The changes in clinical chemistry parameters were not considered to be treatment-related because they were small in magnitude, not dose-related, due to outliers in control animals, and/or were consistent with normal biological variability.

NEUROBEHAVIOUR: No effects were reported related to the battery of functions tested (i.e., changes in the level of motor activity, gait and posture, reactivity to handling or sensory stimuli, grip strength, and stereotypies or bizarre behavior).

ORGAN WEIGHTS: In males given 60 mg Mo/kg bw/day, absolute organ weights were statistically significantly decreased compared to control values for the brain, liver, heart, spleen and pituitary, presumably due to reduced body weight since no significant decrease in any organ weight relative to body weight was observed. A small, but statistically significant, decrease in absolute brain weight was also seen in males at 17 mg Mo/kg bw/day. Brain weight is typically conserved when body weight decreases. However, the decreases in absolute brain weight were not considered biologically significant, because the percent reduction in brain weight (5% at the high dose) was only one-third of the percent reduction in body weight (15% at the high dose), there was no evidence of any histopathological effects in the brain, and a similar effect was not observed in females. Statistically significant decreases in absolute pituitary weight were also observed in males at 5 and 17 mg Mo/kg bw/day but were considered spurious based on the small magnitude of the effect, the absence of dose–response, and a lack of correlative histopathologic findings.

In females, which exhibited less evidence of reduced body weight, there were no significant effects on organ weights at any dose level, with the exception of the thyroid. Females given 5, 17 or 60 mg Mo/kg bw/day had small but statistically significant decreases in absolute and relative thyroid weights; however, these differences were not considered treatment-related since the magnitude of the decreases were small and not dose-related, there was no histopathological correlate, a similar effect was not observed in males, and the concurrent control value was high compared to historical controls.

GROSS PATHOLOGY: There were no macroscopic findings at necropsy related to administration of the test material.

HISTOPATHOLOGY: NON-NEOPLASTIC: Microscopic findings considered possibly to be related to the test substance were limited to changes in the kidneys in females at 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 this finding was present in only two female rats, it is uncommon as a background finding in this age of animal, and therefore it was presumed to be substance-related. Following the 60-day recovery phase, proximal tubule hyperplasia in the kidneys of females administered 60 mg Mo/kg bw/day was not observed in any of the animals.

In males, there were increased incidences of ‘minimal’ and ‘slight’ vacuolation in the cells of the zona fasiculata in the adrenal cortex of males given 5 or 60 mg Mo/kgbw/day. A dose–response relationship was not apparent, and this finding was considered incidental and unrelated to test substance administration. Increased cortical vacuolation of the adrenal is a relatively common background finding in rats that generally reflects normal but variable physiological activity. There were no test substance-related changes in the male or female reproductive tissues.

HISTOPATHOLOGY: NEOPLASTIC (if applicable): Not examined.

HISTORICAL CONTROL DATA (if applicable): No data reported

OTHER FINDINGS:
ESTRUS CYCLES: There were no test substance-related effects on vaginal cytology or estrus cycles when observed during weeks 7–9 of the treatment phase. No significant differences were observed in the mean length of the cycle or the mean number of cycles among groups of females exposed to molybdenum compared to controls (See Table 1 - Other information on results incl. tables). One female with an irregular cycle was observed in the control group and in each treated group.

SPERM EVALUATION: In males, no effect of treatment was observed on spermatid or sperm counts, or on sperm morphology (See Table 2 - Other information on results incl. tables). No significant effect on overall sperm motility was observed at any dose level at the terminal sacrifice, although the 60 mg Mo/kg bw/day males had a slight but statistically significant decrease in progressively motile sperm at the terminal sacrifice (59.0% vs. 69.4% in controls). This slight decrease in progressively motile sperm at the high dose was likely attributable to the control group having a value that approached the upper limit for this parameter among historical control groups (mean of 59.8 ± 16.2%) and was therefore not considered a test substance-related finding. The sperm morphological evaluation found no significant effect in any group on the percent normal sperm or the percent of individual abnormalities (i.e., decapitated, abnormal head, abnormal neck, abnormal tail, midtail blob). At the end of the recovery period, there was no significant effect on any sperm parameter.

ELEMENTAL ANALYSIS OF SERUM, WHOLE BLOOD, LIVER AND KIDNEY: Dietary administration of sodium molybdate dihydrate produced a dose-related increase in molybdenum concentrations in the serum and whole blood (See Table 3 - Other information on results incl. tables). The males had higher serum and/or whole blood levels of molybdenum than the females (23% on average for the three treated groups) at all dose levels at both weeks 4 and 12 of treatment. This difference is not explained by the test substance intake results that showed the females had higher actual intake levels (0, 5.4, 19.0, and 65.2 mg Mo/kg bw/day) compared to males (0, 4.5, 15.1, and 54.8 mg Mo/kg bw/day) at the nominal dose levels of 0, 5, 17, and 60 mg Mo/kg bw/day.

There was no significant accumulation of molybdenum in the serum, since the week 12 serum results were only slightly greater than week 4 serum results. Compared to serum levels, the whole blood levels in week 12 were consistently lower (55% on average for the three treated groups, both sexes) suggesting that the majority of the molybdenum was in the plasma rather than in the red blood cells. During the recovery period, there was a rapid and substantial reduction in serum molybdenum concentrations at Days 2 and 7 after cessation of treatment in the group given 60 mg Mo/kg bw/day.

Dose-related increases in liver and kidney concentrations of molybdenum (dry weight) were observed at the termination of exposure (See Table 3). In contrast to the results in serum and blood, the concentrations of molybdenum in the liver and kidney were higher in females than males at all dose levels. Sixty days after exposure, nearly complete recovery to control molybdenum concentrations was observed in the liver, but not in the kidney.

Consistent with the well-known interaction between molybdenum and copper levels in the body, statistically significant increases in copper concentrations in the serum, whole blood, liver and kidney were observed in both males and females given 60 mg Mo/kg bw/day (See Table 4 - Other information on results incl. tables). In the middle dose group (17 mg Mo/kg bw/day), the levels of copper in the serum in males, and serum, liver and kidney in females were also significantly increased, although to a lesser extent than observed at the high dose. At the low dose (5 mg Mo/kg bw/day), the only statistically significant difference was an increase in the copper concentration in the liver of females.

Dose descriptor:

NOAEL

Effect level:

17 other: mg Mo/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on effects on body weight, body weight gain, food conversion efficiency and renal histopathology (females only) at 60 mg Mo/kg bw/day.

Dose descriptor:

LOAEL

Effect level:

60 other: mg Mo/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on high levels of molybdenum and copper were found in the serum, blood, liver and kidneys of rats treated with 60 mg Mo/kg bw/day.

Dose descriptor:

NOAEL

Remarks:

gonadal, sperm and estrus cycle effects

Effect level:

60 other: mg Mo/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test substance-related effects on gonadal, sperm or estrus cycle were reported.

Critical effects observed:

not specified

Table 1
Length of estrus cycles, number of cycles and number of females with irregular cycles among female rats in the 90 day toxicity study of sodium molybdate dihydrate.^a
Dose (mg Mo/kgbw/day)	Mean length of estrus cycle, days (No. of rats)	Mean number of cycles (No. of rats)	Number of females with irregular cycles
0	4.0 ± 0.11 (19)^b	4.3 ± 0.7 (19)^b	1
5	4.1 ± 0.17 (10)	3.9 ± 0.7 (10)	1
17	4.0 ± 0.26 (10)	4.1 ± 0.9 (10)	1
60	4.1 ± 0.29 (20)	4.0 ± 0.9 (20)	1
^a Daily vaginal smears were taken from each female at approximately the same time each day and stage of estrus determined for 3 weeks starting after completing 6 weeks of treatment.
^b Mean ± standard deviation (n).

Table 2
Mean sperm motility and counts among male rats at the terminal sacrifice in the 90 day toxicity study of sodium molybdate dihydrate (10 animals per group).
Dose (mg Mo/kgbw/day)	Right vas deferens			Right cauda epididymis			Right testis
	Motile sperm (%)	Progressively motile sperm (%)		Weight (g)	Sperm count (millions/g)		Weight (g)	Spermatid count (millions/g)
0 Mean ± SD	97.3	69.4		0.374	542.7		1,710	91.8
	2.6	10.9		0.036	120.5		0.080	21.5
5 Mean ± SD	97.9	60.4		0.399	640.1		1,869	91.4
	2.0	14.4		0.045	97.4		0.169	13.9
17 Mean ± SD	98.0	65.6		0.393	649.0		1,797	89.4
	1.4	8.1		0.038	152.2		0.135	18.7
60 Mean ± SD	98.1	59.0^a		0.374	520.9		1,745	84.7
	1.3	6.8		0.032	132.1		0.132	17.1
^a   Significantly different from the control group (p< 0.05).

Table 3
Molybdenum concentrations (mean ± standard deviation) in serum, whole blood, liver and kidney in rats given sodium molybdate dihydrate in the diet.
Dose (mg Mo/ kgbw/day)	Serum week 4 (ng/mL)	Serum week 12 (ng/mL)	Whole blood week 12 (ng/mL)	Serum Day 2 recovery (ng/mL)	Serum Day 7 recovery (ng/mL)	Liver Terminal sacrifice (lg/g)	Kidney Terminal sacrifice (lg/g)	Liver End of recovery (lg/g)	Kidney end of recovery (lg/g)
Male
n^a	18.72 ± 3.57	19.44 ± 7.18	11.97 ± 1.68	19.10 ± 4.92	20.62 ± 4.89	2.22 ± 0.19	0.93 ± 0.16	1.88 ± 0.40	0.91 ± 0.15
5	1332 ± 379.0	1309 ± 215.5	912.5 ± 212.0	N.D.	N.D.	2.53 ± 0.28	2.30 ± 0.79	N.D.	N.D.
17	4688 ± 616.9	4674 ± 1670	2930 ± 778.3	N.D.	N.D.	4.00 ± 1.23	9.52 ± 6.91	N.D.	N.D.
60	16277 ± 3660	18497 ± 4339	9903 ± 2291	4382 ± 2365	2426 ± 1187	11.49 ± 5.35	41.66 ± 31.41	2.30 ± 0.36	7.04 ± 5.93
Female
n^a	19.86 ± 10.65	17.93 ± 3.47	11.11 ± 2.26	15.14 ± 2.06	33.37 ± 49.15	2.46 ± 0.28	0.94 ± 0.13	2.71 ± 0.18	0.97 ± 0.16
5	991.9 ± 461.3	1121 ± 460.0	720.4 ± 329.1	N.D.	N.D.	3.51 ± 0.45	3.83 ± 2.74	N.D.	N.D.
17	3371 ± 710.4	4312 ± 985.7	2628 ± 479.0	N.D.	N.D.	4.92 ± 0.62	10.94 ± 4.36	N.D.	N.D.
60	13176 ± 3905	15532 ± 5938	7736 ± 2431	6447 ± 5431	2842 ± 2201	12.99 ± 3.65	55.04 ± 27.49	4.58 ± 2.29	16.86 ± 18.29
N.D. = not determined.
^a Group 1 animals received approximately 0.08 mg Mo/kg bw/day from the diet.

Table 4
Copper concentrations (mean ± standard deviation) in serum, whole blood, liver and kidney in rats given sodium molybdate dihydrate in the diet.
Dose (mg Mo/ kgbw/day)	Serum week 4 (ng/mL)	Serum week 12 (ng/mL)	Whole blood week 12 (ng/mL)	Serum Day 2 recovery (ng/mL)	Serum Day 7 recovery (ng/mL)	Liver (lg/g) terminal sacrifice	Kidney (lg/g) terminal sacrifice	Liver (lg/g) end of recovery	Kidney (lg/g) end of recovery
Male
0	1.26 ± 0.29	1.43 ± 0.30	1.12 ± 0.15	1.91 ± 1.22	1.49 ± 0.21	16.96 ± 1.93	30.19 ± 10.59	14.47 ± 2.64	26.31 ± 10.36
5	1.47 ± 0.18	1.62 ± 0.27	1.12 ± 0.14	N.D.	N.D.	19.47 ± 2.97	31.13 ± 6.28	N.D.	N.D.
17	1.74 ± 0.39^b	2.10 ± 0.68^a	1.25 ± 0.28	N.D.	N.D.	18.77 ± 3.00	39.70 ± 13.67	N.D.	N.D.
60	4.51 ± 2.21^b	5.79 ± 2.91^b	2.53 ± 1.21^b	4.14 ± 1.44^b	2.92 ± 0.69^b	26.32 ± 7.55^a	86.04 ± 52.93^a	15.14 ± 1.25	43.39 ± 16.77^a
Female
0	1.77 ± 0.21	1.95 ± 0.23	1.32 ± 0.15	2.03 ± 0.48	1.91 ± 0.19	19.21 ± 2.06	43.54 ± 11.39	19.16 ± 1.78	43.84 ± 19.83
5	1.97 ± 0.26	2.06 ± 0.34	1.34 ± 0.23	N.D.	N.D.	23.85 ± 3.62^a	64.34 ± 24.17	N.D.	N.D.
17	2.12 ± 0.47	2.56 ± 0.72^a	1.40 ± 0.24	N.D.	N.D.	25.16 ± 5.06^a	67.06 ± 23.77^a	N.D.	N.D.
60	4.51 ± 2.17^b	6.63 ± 4.36^b	2.14 ± 0.50^b	7.02 ± 4.16^b	3.56 ± 1.50^b	36.32 ± 7.00^b	138.72 ± 56.34^b	22.34 ± 4.87	74.33 ± 40.12
N.D. = not determined.
^a  Difference from relative control group statistically significantp< 0.05.
^b  Difference from relative control group statistically significantp< 0.001.

Conclusions:

In conclusion, dietary administration of Sodium molybdate dihydrate providing a dose of 60 mg Mo/kg bw/day for 90 days clearly affected body weight gain in male and female rats, and was associated with mild renal effects in two females. Elevated levels of molybdenum were found in the serum, blood, liver and kidneys of rats treated with 60 mg Mo/kg bw/day. Lower doses of 5 and 17 mg Mo/kg bw/day did not cause any treatment-related toxicity. No treatment-related adverse effects on the reproductive organ weights or histopathology, estrus cycles or sperm parameters were observed at any dose level. Thus, the NOAEL for Molybdenum in male and female rats was determined to be 17 mg Mo/kg bw/day based on effects on reduced body weight gain in males and females, and renal effects in two females at 60 mg Mo/kg bw/day. The NOAEL for gonadal, sperm and estrus cycle effects was 60 mg Mo/kg bw/day.

Executive summary:

In a 90-day subchronic toxicity study (OECD 408) Sodium molybdate dihydrate (>99% purity) was administered to 20 animals/sex/group (for control and high dose groups) and 10 animals/sex/dose (for low and middle dose groups) to Crl:CD(SD)IGS BR, VAF/ Plus Sprague–Dawley derived Rats in the diet at nominal dose levels of 0, 5, 17, 60 mg Mo/kg bw/day.

The NOAEL was 17 mg Mo/kg bw/day, based on effects on body weight, body weight gain, food conversion efficiency and renal histopathology (females only) at 60 mg Mo/kg bw/day. No treatment-related adverse effects on reproductive organ weights or histopathology, estrus cycles or sperm parameters were observed at any dose level.

This subchronic toxicity study in the rat is acceptable and satisfies the guideline requirement for a subchronic oral study OECD 408 in rats. 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

17 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No data is available for Dimolybdenum carbide (target substance). Thus, available data from Sodium molybdate was used in a read-across approach. Due to a lower water solubility of Dimolybdenum carbide compared to Sodium molybdate the resulting bioavailability (toxicity potential) would also be expected to be lower. Therefore, the read across to the source substance Sodium molybdate is adequately protective. Details on the read-across rational are provided in section 13.

A repeated subchronic oral dose toxicity study [OECD 408] was conducted using male and female Wistar rats at doses of 0, 5, 17, and 60 mg Mo/kg/day of Sodium molybdate dihydrate (>99% w/w). The animals received the test item daily over a period of 90 days via the drinking water. At 60 mg Mo/kg/bw effects on effects on body weight, body weight gain, food conversion efficiency and light diffuse hyperplasia of the proximal tubules in the kidney of two females were observed. Based on the results reported the NOAEL for orally administered Sodium molybdate dihydrate is considered to be 17 mg Mo/kg bw/day for both sexes. Based on the NOAEL for administered molybdenum the NOAEL for the target substance Dimolybdenum carbide can be calculated with 18 mg/kg bw/day (not accounting for differences in solubility/bioavailability).

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

GLP guideline study conducted with Sodium molybdate.

Justification for classification or non-classification

Based on the available data from the read-across partner Sodium molybdate dihydrate, Dimolybdenum carbide does not warrant classification for specific target organ toxicity in accordance to CLP as no severe toxic effects were observed.