Calcium molybdate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Effect levels expressed in terms of the test item sodium molybdate dihydrate ("SMD") and in terms of the element molybdenum (Mo):

The NOAEL for reproductive toxicity is > 100 mg SMD/kg bw/day (> 40 mg Mo/kgbw/day), based on an OECD Test Guideline 416 multigenerational study (Hoberman, 2017). This is supported by the absence of any adverse effects on parameters relating to fertility at up to 150 mg SMD/kg bw/day (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

Reference 1

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-07-07 to 2017-03-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

2001-01-22

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: controlled room temperature

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The Sprague Dawley rat was chosen as the animal model for this study because: 1) it is an accepted rodent species for preclinical toxicity testing by regulatory agencies; 2) this species and strain has been demonstrated to be sensitive to developmental toxicants; and 3) historical data and experience exist at the testing facility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC, United States
- Age upon arrival at laboratory: males: approx. 45 days; females : approx. 37 days
- Weight upon arrival at laboratory: males: 150 - 196 g; females: 77 - 108 g
- Weight at randomization and study assignment: males: 174 - 211 g; females: 91 - 120 g
- Housing: individually housed in solid-bottomed cages, except during the cohabitation and postpartum periods and during urine collection; during cohabitation: each pair of rats was housed in the male rat’s nesting box; during the postpartum period: each dam and delivered litter were housed in individual nesting boxes until weaning; duration of urine collection: rats selected for collection were housed in a stainless steel, wire bottom metabolism cage; Nesting material (Bed-o'Cobs®) was provided.
- Diet (ad libitum): Certified Rodent Diet® #5002 (PMI® Nutrition International)
- Water (ad libitum): during the acclimation period: water was passed through a reverse osmosis membrane and chlorine was added; during the exposure period: R.O. deionized water without chlorine
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19°C to 25°C)
- Relative humidity: 30% to 70%
- Air changes (per hr): ≥ 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF TEST SUBSTANCE
- test substance dosing formulations were prepared based on sponsor instructions and a previous range-finding study (please refer to Section 7.8.1 Toxicity to reproduction: s_Hoberman_2016_dose-range finder (diet)) at appropriate concentrations to meet dose level requirements.
- the formulations prepared in powdered diet were prepared as needed based on 42-day stability and stored at ambient temperature until use
- a concentration of the test and/or control substance in the diet was offered to the rats, and the mg/kg/day doses consumed were calculated and presented for periods corresponding to body weight and food consumption observations

Details on mating procedure:

- M/F ratio per cage: 1 male / 1 female
- Length of cohabitation: maximum of 10 days
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as gestation day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individual housing
- Female rats not mated after completion of the 10-day cohabitation period were considered to be at gestation day 0 on the last day of cohabitation.

F1 generation
- M/F ratio per cage: 1 male / 1 female
- Length of cohabitation: maximum of 17 days
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as gestation day 0 of pregnancy
- After 10 days of unsuccessful pairing replacement of first male by another male and remained in cohabitation for a maximum of 7 additional days.
- After successful mating each pregnant female was caged (how): individual housing
- Female rats not mated after completion of the 17-day cohabitation period were considered to be at gestation day 0 on the last day of cohabitation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for analysis of concentration of molybdenum according to the following schedule:
- first preparation
- at approximately monthly intervals
- last preparation
Analyses described below were performed by inductively coupled plasma - mass spectroscopy (ICP-MS).

Analysis of concentration of molybdenum:
- duplicate middle sets of samples for each sampling time point were collected.
- concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% of theoretical concentration.
- each individual sample concentration result was considered acceptable if it was within or equal to ± 20%.

Analysis of stability:
Stability analyses performed previously in a dose range finder study (please refer to section 7.8.1 Toxicity to reproduction: s_Hoberman_2016_dose-range finder (diet)) demonstrated that the test substance is stable in the diet when prepared and stored under the same conditions at concentrations as those used in the present study (did not cover the high concentration of diet at 2450 ppm used in the current study).

Results:
- all diet formulations that were analyzed were found to be within or very close to the planned quality control parameters set forth in the protocol, and thus appropriate for use.
- the amount of test substance used each week to prepare dosed diet was adjusted weekly based on estimated average body weight for the next week of study, to achieve a daily exposure of 40 mg Mo/kg bw/day from the diet. This weekly adjusting of concentration was continued throughout the premating period for both males and females, and continued for the males until termination. During the gestation and lactation periods for the female rats, test diet was adjusted weekly based on the day of gestation or lactation to provide test diets with concentrations that would achieve the 0, 5, 17 or 40 mg Mo/kg bw/day dosesfrom sodium molybdate dihydrate.
- homogeneity and stability of the test diets for 14, 28 and 42 days at room temperature and frozen was established during the conduct of the range finder study (please refer to section 7.8.1 Toxicity to reproduction: s_Hoberman_2016_dose-range finder (diet)).

- concentration analyses: all analyzed samples were within or equal to the acceptance criteria of ± 15% for the diet (individual sample within or equal to ± 20% for the diet) of their theoretical concentrations.

Duration of treatment / exposure:

Parental (P) generation:
- males: at least 10 weeks before cohabitation, during the cohabitation, and continued through to the day before euthanasia
- females: at least 10 weeks before cohabitation, during the cohabitation, gestation, littering and lactation periods and continuing through to the day of euthanasia

F1 generation:
- pups were directly exposed after they begin consuming food/water (approx. on Day 14 postpartum).
- - pups will have been exposed during maternal gestation (in utero exposure) or via maternal milk during the lactation period (Ref Karolina Kot et al 2019)
- weaned pups were directly exposed at least 10 weeks before cohabitation, during the cohabitation, gestation, littering and lactation period and continuing through to the day of euthanasia.

F2 generation:
- pups were directly exposed after they begin consuming food/water (approx. on Day 14 postpartum).
- pups will have been exposed during maternal gestation (in utero exposure) or via maternal milk during the lactation period (Ref Karolina Kot et al 2019)

Karolina Kot, et al, 2019, International Journal of Environmental Research and Public Health. Interactions between 14 Elements in the Human Placenta, Fetal Membrane and Umbilical Cord. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6540151/

Frequency of treatment:

ad libitum

Details on study schedule:

- Selection of parents from F1 generation when pups were 21 days of age (weaning day; at least one male pup and one female pup per litter were selected; selection of runts, or pups otherwise impaire, was avoided).

- F1 parental animals not mated until 10 weeks after selected from the F1 litters.

- Age at mating of the mated animals (F1 generation) in the study: approx. 90 days of age

Dose / conc.:

100 mg/kg bw/day

Remarks:

equivalent to 40 mg Mo/kg bw/day

No. of animals per sex per dose:

24 males / 24 females

Control animals:

yes, plain diet

Details on study design:

The main driver for conducting this 2-generation study was for regulatory purposes in the United States of America. In the past, regulations deriving health thresholds for molybdenum have often relied on the non-guideline study by Fungwe et al. (1990) in which rats were exposed to molybdenum via drinking water, and US EPA were seeking a more robust study. Therefore, this 2-generation study was also conducted via drinking water, to see if it would be possible to replicate the findings reported by Fungwe et al. (1990). The range-finder and the main study each included three dose levels via drinking water. Since for humans the exposure via the diet is the more relevant route of exposure, the range-finder also included 3 dose levels via the diet. For the main study, only the top-dose was administered via the diet. The US EPA Office of Water was involved in the decision to select drinking water as the main route of administration in the main study.
For technical reasons, four separate study records had to be prepared in IUCLID for the drinking water and diet parts of the range-finder and main study, respectively. All parts of the study should be considered together.

This study record is for the main study, dietary administration part of the study.


The dose levels were selected based on the results of previous sub-chronic and developmental toxicity studies conducted by the Sponsor, and a rangefinder study (please refer to Section 7.8.1 Toxicity to reproduction: s_Hoberman_2016_dose-range finder) in which clear effects were observed in the bodyweights at the top dose levels (40 mg Mo/kgbw/day). In the sub-chronic (90 day study) in rodents a top dose of 60 mg Mo/kgbw/day produced an excessive bodyweight loss in males, and renal toxicity in females (Murray et al., 2014)*. In the rangefinder study, doses as high as 40 Mo/kgbw/day were chosen and administered in the diet or water. The results of the rangefinder study are summarized below:
No deaths related to sodium molybdate dihydrate occurred in the rangefinder study (please refer to Section 7.8.1 Toxicity to reproduction: s_Hoberman_2016_dose-range finder). Administration of sodium molybdate dihydrate in the water or diet reduced body weight gains, body weights, water and feed consumption in the 40 mg Mo/kg bw/day exposure group. Within the intervals evaluated (premating, gestation, lactation) sporadic reductions in body weight, feed and water consumption occurred in the 3 and 20 mg Mo/kg bw/day exposure groups, and these reductions were not considered adverse.
Terminal body weights in the male rats were reduced in both the diet and water 40 mg Mo/kg bw/day exposure groups in the rangefinder. In the groups exposed via the diet, the absolute weight of the seminal vesicles with fluid was significantly reduced in the 20 and 40 mg Mo/kg bw/day dose groups compared to the control group value. The ratio of the weight of the left and right testes and the left cauda epididymis to the terminal body weight in the 40 mg Mo/kg bw/day diet dose group were all significant increased compared to the control group values.
The absolute weight of the seminal vesicles with fluid and the weight of the left kidney were significantly reduced in the 40 mg Mo/kg bw/day water dose groups compared to the control group value in the rangefinder. The ratio of the weight of the left and right testes to the terminal body weight in the 40 mg Mo/kg bw/day water dose group were both significantly increased compared to the control group values.
The changes in organ weights were not considered adverse in the rangefinder as there were no histological changes in any organ evaluated and the increased organ to body weight ratios indicated that the changes reflected the reduced terminal body weights.
Oestrous cycling, mating and fertility were not affected by exposures as high as 40 mg Mo/kg bw/day in the diet or water in the rangefinder. Sperm parameters were comparable among the diet and water exposure groups. A reduced number of females pregnant (6/10 pregnant) in the 40 mg Mo/kg bw/day water exposure group was observed. The litter size and survival to weaning of the F1 generation was not affected by maternal doses of sodium molybdate dihydrate in the diet or water.

Based on the results of this study in diet and the drinking water study (separate IUCLID RSS) doses
as high as 40 mg Mo/kg bw/day were selected for the evaluation in the full multigenerational study since this resulted in a reduction in body weights in the range finder in excess of 10% in the males which is generally accepted as a maximum tolerated dose level and in the a comparator group, at 40 mg Mo/kg bw/day the diet was also included in the study design to evaluate potential carrier differences. In addition, in females, in the diet administration range finder study, body weights were significantly reduced to 90.1% of controls prior to cohabitation and also significantly reduced during gestation and lactation
Exposure through the drinking water was used because of the apparent reduced number of females (6/10) in the 40 mg Mo/kg bw/day water exposure group in the rangefinder and in addition to assist in trying to replicate the findings reported by Fungwe et al. (1990) as agreed with the USA EPA (see first paragraph of this section) .

*Reference:
- Murray FJ, Sullivan FM, Tiwary AK, Carey S. 90-Day subchronic toxicity study of sodium molybdate dihydrate in rats. Regulatory Toxicology and Pharmacology 2014; 70:579-588.

Positive control:

none

Parental animals: Observations and examinations:

1) PARENTAL (P) GENERATION / F1 GENERATION (producing F2 generation) Recorded as PARENTAL (P) GENERATION - P0 and F1 GENERATION (producing F2 generation) -P1

CAGE SIDE OBSERVATIONS: Yes
Time schedule:
- general health/ mortality and moribundity: twice daily, once in the morning and once in the afternoon.
- maternal observations: apparently pregnant females were observed frequently from the expected day of parturition to determine, where possible, onset and duration of parturition; thereafter, maternal observations were recorded at least once daily. Observed abnormal behaviour was recorded daily

DETAILED CLINICAL OBSERVATIONS: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily during exposure periods and prior to euthanasia
- during cohabitation, when two rats occupied the same nesting box with one water bottle (using the lower concentration available at the time, as applicable), replenishment of the water was documented
- water consumption was not tabulated after lactation day 14, when it was expected that pups would begin to consume water.

FURTHER OBSERVATIONS:
- urine collection: urine samples were collected approx. 24 hours on the following days:
P Generation (12 rats/group/sex):
males: study days 30, 31, 32 or 33 and on study days 106, 108, 109 or 110
females: study days 30, 31, 32 or 33, gestation day 30 or lactation days 25, 29, 30, 31, 32, 33, 34, 35 or 36
F1 Generation (12 rats/group/sex)
males: days 48, 49, 50 or 51 postpartum, and on days 144, 145 or 146 postpartum.
females: days 48, 49, 50 or 51 postpartum and gestation days 47or 48 or DL 23, 24, 25, 26, 27 or 28.
The urine samples were analysed for concentrations of molybdenum and other elements.

-blood collection: blood was collected on the day before scheduled euthanasia (collection: morning and afternoon; 12 rats/group/sex (same animals selected urine collection)).
Serum samples were analysed for concentrations of molybdenum and other elements.

- mating performance: mating was evaluated daily during the cohabitation period and until confirmed by spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ.
- natural delivery observations: female rats were evaluated for adverse clinical signs (including any indication of prolonged parturition) and duration of gestation (gestation day 0 until the time the first pup was observed).

2) PARENTAL (P) GENERATION - Po

CAGE SIDE OBSERVATIONS: Yes
Time schedule:
- general appearance: at least weekly during the acclimation period, on the day of randomization, daily during the dose period, and on the day of scheduled euthanasia

BODY WEIGHT: Yes
Time schedule for examinations:
- males: individually weighed on the day after arrival, at least weekly during the acclimation period, at least weekly during the dose period and on the day of scheduled euthanasia.
- females: individually weighed on the day after arrival, at least weekly during the acclimation period, at least weekly during the dose period on gestation days 0, 7, 10, 14, 20, 25, and weekly thereafter (as applicable) lactation days 0, 4, 7, 14, 21, 28, 35, 42, 49, 56 and 58 and the day of scheduled euthanasia

FOOD CONSUMPTION: Yes
- males: at least weekly during the dose period
- females: at least weekly during the dose period on gestation days 0, 7, 10, 14, 18, 20 and 25 and lactation days 0, 4, 7, 10, 14, 21, 28, 35, 42, 49, 56 and 58, and on the day of scheduled euthanasia.
- during cohabitation, when two rats occupied the same nesting box with one food jar (using the lower concentration available at the time, as applicable), replenishment of the food jars was documented.
- food consumption was not tabulated after lactation day 14, when it was expected that pups would begin to consume maternal food.

FURTHER OBSERVATIONS:
- duration of gestation: duration of gestation was calculated from gestation day 0 to the day the first pup was observed.
- parturition: apparently pregnant females were observed frequently from the expected day of parturition to determine, where possible, onset and duration of parturition.
- number of implantation sites

3) F1 GENERATION (producing F2 generation) - P1

CAGE SIDE OBSERVATIONS: Yes
Time schedule:
- general appearance:
males: at least weekly and on the day of scheduled euthanasia
females: at least weekly, on gestation days 0, 7, 10, 14, and 20, and lactation days 0, 4, 7, 14, and 21, and the day of scheduled euthanasia

BODY WEIGHT: Yes
Time schedule for examinations:
- males: individually weighed at least weekly and on the day of scheduled euthanasia.
- females: individually weighed at least weekly, on gestation days 0, 7, 10, 14, 20, and 25 (as applicable) and lactation days 0, 4, 7, 14, and 21, at least weekly thereafter, and on the day of scheduled euthanasia

FOOD CONSUMPTION: Yes
- males: at least weekly during the exposure period
- females: at least weekly during the exposure period, on gestation days 0, 7, 10, 14, 18 and 20, and lactation days 0, 4, 7, 10 and 14.
- during cohabitation, when two rats occupied the same nesting box with one food jar, replenishment of the food jars was documented.
- food consumption was not tabulated after lactation day 14, when it was expected that pups would begin to consume maternal food.

SEXUAL MATURATION
- sexual maturation was evaluated daily until the criterion was achieved.
- females: observation for vaginal opening began on Day 27 postpartum.
- males: observation for preputial separation began on Day 34 postpartum.
- body weights were recorded on the day the criteria was achieved.

Oestrous cyclicity (parental animals):

Parental (P) generation (P0) and F1 generation (producing the F2 generation) (P1) :
Oestrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Efforts to avoid inducing false pregnancy signs were performed during the daily procedure. Samples were collected for 14 consecutive days before pairing, during the cohabitation period until spermatozoa are observed in a smear of the vaginal contents and/or a copulatory plug is observed in situ, and on the day of scheduled euthanasia.

Sperm parameters (parental animals):

Parameters examined in P (P0) and F1 male generations (P1):
- sperm motility (sperm from each vas deferens)
- sperm concentration in left cauda epididymis (sperm per gram of tissue weight)
- sperm morphology in portion of left cauda epididymides (determination of the percentage of normal sperm in a sample of at least 200, where feasible; and qualitative evaluation of abnormal sperm)
- spermatid counts (left testis; spermatids/gram of tissue weight)

Litter observations:

STANDARDISATION OF LITTERS (F1 and F2 generations)
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring:
- pup weight (F1 generation: days 0 (birth), 4, 7, 14 and 21 postpartum; F2 generation: days 0 (birth), 4, 7, 14, 21, 22, 23, and 24 postpartum)
- litter size
- viability at birth (number of live and dead pups at birth)
- postnatal mortality (at least twice daily during the preweaning period)
- number of pups in each litter (once daily during the preweaning period)
- clinical observations (daily during the preweaning period)
- developmental landmarks (pinna unfolding (day initated: day 2 postpartum), hair growth (day initated: day 7 postpartum), incisor eruption (day initated: day 9 postpartum), eye opening (day initated: day 12 postpartum), anogenital distance (once on day 4 postpartum (F2 only)); testing continued until the day the criterion was attained by all pups in the litter or until the day of scheduled euthanasia.)
- clinical chemistry: blood samples were collected from 2 pups/sex/litter (when possible) on Day 23 ± 2 days postpartum from the F1 and F2 generation pups not selected for continued observation, as applicable. Serum samples were analysed for concentrations of molybdenum and other elements.

GROSS EXAMINATION OF DEAD PUPS (F1 and F2 generation):
- Day 0 to 21 postpartum: pups that were found dead before examination of the litter for pup viability (Day 0 postpartum) were evaluated for vital status at birth. The lungs were removed and immersed in water. Pups with lungs that sank were identified as stillborn; pups with lungs that floated were identified as liveborn and to have died shortly after birth.
Pups that died (Days 1 to 21 postpartum) or were euthanized (Days 0 to 21 postpartum) before scheduled termination were examined for gross lesions and the cause of death or condition as soon as possible after the observation was made. The presence or absence of milk in the stomach was determined.

Postmortem examinations (parental animals):

1) PARENTAL (P) GENERATION (P0)
SACRIFICE
- Male animals: study days 147 to 151
- Maternal animals: lactation days 58 to 64
Animals that died or were euthanized before scheduled termination were examined for the cause of death or condition as soon as possible after the observation was made. The rats were examined for gross lesions, and a gross necropsy of the thoracic, abdominal, and pelvic viscera was performed.
Pregnancy status and uterine contents of female rats were recorded. Conceptuses in utero were examined to the extent possible as appropriate.

OVARIAN AND UTERINE EXAMINATIONS
- reproductive tract was dissected from the abdominal cavity.
- number and distribution of implantation sites were recorded.
- uteri of apparently nonpregnant rats were examined to confirm the absence of implantation sites

GROSS NECROPSY
- a gross necropsy of the thoracic, abdominal and pelvic viscera was performed at scheduled euthanasia and for all rats that were found dead or euthanized prior to scheduled termination.

ORGAN WEIGHTS
- the following organs were weighed at necropsy for all scheduled euthanasia animals: brain, epididymis, left cauda of epididymis, adrenal gland, coagulating gland, pituitary gland, prostate, seminal vesicles, thyroid, kidney, liver, ovaries, oviduct, spleen, thymus, testes, uterus
- organ weights were not recorded for animals found dead or euthanized in poor condition or in extremis.
- paired organs were weighed together, unless otherwise noted.
- organ weight as a percent of body weight (using the terminal body weight) was calculated.

HISTOPATHOLOGY
- the following representative samples of the tissues were collected from all animals and preserved: brain, cervix, epididymis, left cauda of epididymis, adrenal gland, coagulating gland, parathyroid, pituitary gland, prostate, seminal vesicles, thyroid, gross lesions (masses), kidney, liver, lungs, ovaries, oviduct, spleen, thymus, testes, uterus
- the following tissues were collected, processed, embedded, sectioned, and stained for microscopic evaluation: cervix, epididymis, left cauda of epididymis, adrenal gland, coagulating gland, parathyroid, pituitary gland, prostate, seminal vesicles, thyroid, gross lesions (masses), kidney, lungs, ovaries, oviduct, testes, and uterus
- entire left and right ovaries were embedded in separate paraffin blocks. Each ovary was sectioned beginning at 200 microns within the ovary. The first section was collected and designated as section 1 for follicle counts. After advancing the microtome 100 microns, the next section was collected as section 2. This process continued until a total of five sections from each ovary were collected for follicle counts, plus one section collected (between follicle count sections 3 and 4) on a separate slide for routine histopathological evaluation. The five sections were placed on one slide for each ovary. Section 1 was placed closest to the slide label, continuing in a linear pattern,
until five sections were on the same slide (section 5 being most distal to the slide label).
- tissues processed for primordial follicles were retained for possible future analysis. Presence or absence of corpora lutea was recorded for each animal. Group means and standard deviations were calculated for each animal (left, right, and both ovaries combined). Where the sample size was appropriate (N> 2), group mean values were compared in order to detect any significant differences in the number of primordial follicles in left ovaries, right ovaries, and both ovaries combined. Treated groups evaluated were compared statistically against groups that received control article only. Data were subjected to the Kruskal-Wallis nonparametric statistical test. If a significant difference was determined at p< 0.05, data were further analyzed using the Wilcoxon (Mann-Whitney U) Test.

TISSUE ANALYSIS
- at euthanasia, the liver and right kidney were collected
- tissues were analysed for concentrations of molybdenum and other elements

2) F1 GENERATION (producing the F2 generation) (P1)

SACRIFICE
- Male animals: study days 159 to 164
- Maternal animals: lactation days 24 to 34
Postweaning (> Day 21 postpartum): the F1 generation animals which died or were euthanised before before scheduled termination were examined for the cause of death or condition as soon as possible after the observation was made. The rats were examined for gross lesions, and a gross necropsy of the thoracic, abdominal, and pelvic viscera was performed.
Pregnancy status and uterine contents of the female rat were recorded. Conceptuses in utero were examined to the extent possible, where applicable.

OVARIAN AND UTERINE EXAMINATIONS
- reproductive tract was dissected from the abdominal cavity.
- number and distribution of implantation sites were recorded.
- uteri of apparently nonpregnant rats were examined to confirm the absence of implantation sites

GROSS NECROPSY
- a gross necropsy of the thoracic, abdominal and pelvic viscera was performed at scheduled euthanasia and for all rats that were euthanized prior to scheduled termination.

ORGAN WEIGHTS
- brain, spleen, and thymus were weighed at necropsy from one arbitrarily selected pup not selected for further evaluation per sex/litter at scheduled euthanasia, when possible.
- the following organs were weighed at necropsy for all scheduled euthanasia animals: brain, epididymis, left cauda of epididymis, adrenal gland, pituitary, prostate, seminal vesicles, thyroid, kidney, liver, ovaries, spleen, thymus, testes, and uterus
- organ weights were not recorded for animals euthanized in poor condition or in extremis.
- paired organs were weighed together, unless otherwise noted.
- organ weight as a percent of body weight (using the terminal body weight) was calculated.

HISTOPATHOLOGY
- the following representative samples of the tissues were collected from all animals and preserved: brain, cervix, epididymis, left cauda of epididymis, adrenal gland, parathyroid, pituitary gland, prostate, seminal vesicles, thyroid, gross lesions (masses), kidney, liver, lungs, ovaries, oviduct, spleen, thymus, testes, and uterus
- one control group animal/sex was selected from which all tissues examined at necropsy were retained, in order to provide control tissues for any possible histopathological evaluations of gross lesions.
- the following tissues were collected, processed, embedded, sectioned, and stained for microscopic evaluation: brain, cervix, epididymis, left cauda of epididymis, adrenal gland, parathyroid, pituitary gland, prostate, seminal vesicles, thyroid, gross lesions (masses), kidney, lungs, ovaries, oviduct, spleen, testes, and uterus
- entire left and right ovaries were embedded in separate paraffin blocks. Each ovary was sectioned beginning at 200 microns within the ovary. The first section was collected and designated as section 1 for follicle counts. After advancing the microtome 100 microns, the next section was collected as section 2. This process continued until a total of five sections from each ovary were collected for follicle counts, plus one section collected (between follicle count sections 3 and 4) on a separate slide for routine histopathological evaluation. The five sections were placed on one slide for each ovary. Section 1 was placed closest to the slide label, continuing in a linear pattern, until five sections were on the same slide (section 5 being most distal to the slide label).
- tissues processed for primordial follicles were retained for possible future analysis. Presence or absence of corpora lutea was recorded for each animal. Group means and standard deviations were calculated for each animal (left, right, and both ovaries combined). Where the sample size was appropriate (N> 2), group mean values were compared in order to detect any significant differences in the number of primordial follicles in left ovaries, right ovaries, and both ovaries combined. Treated groups evaluated were compared statistically against groups that received control article only. Data were subjected to the Kruskal-Wallis nonparametric statistical test. If a significant difference was determined at p< 0.05, data were further analyzed using the Wilcoxon (Mann-Whitney U) Test.

Postmortem examinations (offspring):

1) F1 GENERATION (pups not selected for continued observation)
SACRIFICE / GROSS NECROPSY
- all pups not selected for continued observation on Day 24 ± 2 days postpartum were euthanized and examined for gross lesions
- gross lesions were preserved.
- for one randomly-selected pup/sex/litter, the brain, spleen, thymus, thyroid, ovaries and uterus or testes and epididymides were preserved for possible future analysis.
- carcasses were discarded without further evaluation.

HISTOPATHOLOGY (pups not selected for continued observation)
The following tissues were identified for microscopic evaluation (target tissues) from 1 pup/sex/litter (when possible): brain, cervix, epididymis, left cauda of epididymis, adrenal gland, parathyroid, pituitary gland, prostate, seminal vesicles, thyroid, gross lesions (masses), kidney, lungs, ovaries, oviduct, spleen, testes, and uterus

2) F2 GENERATION
SACRIFICE / GROSS NECROPSY / ORGAN WEIGHTS
- on Day 24 ± 2 days postpartum, a gross necropsy of the thoracic, abdominal and pelvic viscera was performed for all pups.
- pups with gross lesions were preserved
- in addition, the following tissues were weighed/retained for 1 pup/sex/litter (when possible): brain, epididymis, adrenal gland, pituitary gland, prostate, seminal vesicles, thyroid, kidney, liver, ovaries, spleen, thymus, testes, and uterus
- organ weights were not recorded for animals euthanized in poor condition or in extremis.
- paired organs were weighed together, unless otherwise noted.
- all other tissues/carcasses were discarded

HISTOPATHOLOGY
- samples of the following tissues were collected from 1 arbitrarily selected pup/sex/litter (when possible) and all pups with gross lesions and preserved: brain, cervix, epididymis, adrenal gland, pituitary gland, prostate, seminal vesicles, thyroid, gross lesions/ masses, kidney, liver, lungs, ovaries, oviduct, spleen, thymus, testes, and uterus
- tissues identified for microscopic evaluation (target tissues) were examined from all pups with gross lesions: cervix, epididymis, adrenal gland, pituitary gland, prostate, seminal vesicles, thyroid, gross lesions/ masses, kidney, lungs, ovaries, oviduct, testes, and uterus

3) F1 GENERATION / F2 GENERATION
TISSUE ANALYSIS
- at euthanasia, the liver and right kidney were collected
- tissues were analysed for concentrations of molybdenum and other elements

Statistics:

Means, SD & % were calculated, as appropriate. Adult data were evaluated with the individual rat as the unit measured. Litter values were used in evaluation of pup data, as appropriate.
Clinical observation incidence data, & other proportional data were analyzed as contingency tables using the Fisher's Exact Test (1)*.
Continuous data, incl. variables with interval or ratio scales of measurement, such as body weights, food consumption & % motility were analyzed as follows:
Bartlett's Test of Homogeneity of Variances (2)* was used to test the hypothesis that all dose groups had equal variance. A nonsig. result (p>0.001) indicated that an assumption of homogeneity of variance was appropriate, & the data were compared using the ANOVA (3)*. If that test was sig. (p≤ 0.05), the groups given the test substance were compared with the control group using Dunnett's Test (4)*. If Bartlett's Test was sig. (p≤ 0.001), the ANOVA was not appropriate, & the data were analyzed as follows:
The Kruskal-Wallis Test was used to analyze the data, & in the event of a sig. result (p≤ 0.05), Dunn's Test (5)* was used to compare the groups given the test item with the control group. Count data, such as days in cohabitation, day a developmental landmark appears or No. of implantation sites, were analyzed using the Kruskal-Wallis Test & in the event of a sig. result (p≤ 0.05), Dunn's Test was used to compare the groups given the test item with the control group.
*References:
1) Siegel S. Nonparametric statistics for the behavioral sciences. New York (NY): McGraw-Hill Co; 1956. p. 96-105.
2) Sokal RR, Rohlf FJ. Biometry: the principles and practice of statistics in biological research. San Francisco (CA): Freeman & Co; 1969. p. 370-1.
3) Snedecor GW, Cochran WG. Statistical methods. 6th Ed. Iowa State University Press, Ames; 1967. p. 258-98.
4) Dunnett CW. J Am Stat Assoc 1955;50:1096-121.
5) Dunn OJ. Technometrics 1964;6(3):241-52.

Reproductive indices:

- mating index: percentage of pairings that resulted in matings
- fertility Index: percentage of matings that resulted in pregnancies
- gestation Index: Percentage of pregnancies that resulted in birth of live litters

Offspring viability indices:

- live birth index: percentage of pups born alive
- viability Indices: percentage of pups alive day 0 postpartum that survived to 3 days postpartum
- lactation Index: percentage of pups alive day 4 that survived 21 days postpartum

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Five male rats at 40 mg Mo/kg bw/day in the diet had vocalization to touch. However any toxicological significance is unclear

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1) Males - 40 mg Mo/kg bw/day: body weights were reduced or significantly reduced (p≤ 0.01) compared to the concurrent control group for each weekly interval from study days 22 to 71 (last weight prior to cohabitation). On study day 71, body weight was 94.1% of the control group value. Body weights continued to be reduced or significantly reduced (p≤ 0.05 to p≤ 0.01) post cohabitation until euthanasia
- 40 mg Mo/kg bw/day: body weight gains compared to the concurrent control group were reduced and/or significantly reduced (p≤ 0.01) for study days 1 to 71 and study days 1 to 143, with the exception on study days 57 to 64 when a significant increase (p≤ 0.01) occurred.

2) Females - no significant changes during pre-mating . Gestation body weights were significantly reduced (p≤ 0.05 to p≤ 0.01) for gestation days 7, 10 and 14 compared to the concurrent control group values. Body weight gains were significantly reduced (p≤ 0.05) for gestation days 0 to 7. Lactation body weights were reduced or significantly reduced (p≤ 0.05 to p≤ 0.01) for all days of lactation evaluated from lactation days 0 to 56. Lactation body weight gains did not differ significantly from the control group values for any interval evaluated.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Effects that are considered as adverse and/or treatment related are described in the individual fields above.
Effects that are considered as non-adverse and/or not treatment-related are described in this field "Details on results".

CLINICAL SIGNS
1) males
- 40 mg Mo/kg bw/day: five rats in this group had vocalization to touch (not statistically significantly increased compared to control group). The toxcological significance is unclear.
- all other clinical observations were considered unrelated to the test substance (observations occurred in only one to three rats; and/or observation is common in this species and strain), which included: torn pinna, soft or liquid faeces, ungroomed coat, chromorhinorrhea, a scab, chromodacryorrhea and abrasion.

2) females
- 40 mg Mo/kg bw/day: no adverse clinical observations related to the test substance occurred during the premating, gestation or lactation periods.
- all clinical observations were considered unrelated to the test substance (observations occurred in only one rat; and/or observation is common in this species and strain), which included: sparse hair coat, swollen and/or torn pinna, hunched posture, chromorhinorrhea, chromodacryorrhea, swollen snout and misaligned incisors.

MORTALITY
- 40 mg Mo/kg bw/day: no deaths considered related to sodium molybdate dihydrate occurred in the P generation.

Control group: one female rat was euthanized due to adverse clinical observations on day 23 of lactation. These clinical observations all occurred on lactation day 23 and included limited use of the right forelimb (limb with swelling in the axillary region), mild dehydration, hunched posture and thin body condition. The rat was apparently injured on lactation day 23. Body weight, food and water consumption during lactation demonstrated normal fluctuations. This dam had a normal litter of 18 pups all of which survived to weaning. There was a small thymus at necropsy that correlated, microscopically, with mild decreased cellularity. Additional microscopic findings including minimal infiltrates of histiocytes in the lung and mild pigmented macrophages in the uterus were considered incidental. The swelling in the right axillary region correlated, microscopically, with neutrophilic inflammation in the mammary gland tissue and the small thymus seen at necropsy correlated with mild decreased cellularity.
- 40 mg Mo/kg bw/day: one female rat was euthanized due to adverse clinical observations on lactation day 43. These clinical observations all occurred on lactation day 43 and included a swollen snout, misaligned incisors, chromorhinorrhea and chromodacryorrhea. The rat was apparently injured on lactation day 43. Body weight, food and water consumption during lactation demonstrated normal fluctuations. This dam had a normal litter of 14 pups all of which except for one pup survived to weaning. A fractured palate was found at necropsy. An additional microscopic finding was mild pigmented macrophages in the uterus that was considered incidental.

BODY WEIGHT AND WEIGHT CHANGES
1) males
- 40 mg Mo/kg bw/day: body weights were reduced or significantly reduced (p≤ 0.01) compared to the concurrent control group for each weekly interval from study days 22 to 71 (last weight prior to cohabitation). On study day 71, body weight was 94.1% of the control group value. Body weights continued to be reduced or significantly reduced (p≤ 0.05 to p≤ 0.01) post cohabitation until euthanasia with significant reductions (p≤ 0.05 to p≤ 0.01) on study days 129, 136 and 143. On study day 143 body weight was 91.4% of the control group value.
- 40 mg Mo/kg bw/day: body weight gains compared to the concurrent control group were reduced and/or significantly reduced (p≤ 0.01) for study days 1 to 71 and study days 1 to 143, with the exception on study days 57 to 64 when a significant increase (p≤ 0.01) occurred.

2) females
- body weights and body weight gains were comparable among the groups for the pre-mating period.
- 40 mg Mo/kg bw/day: On study day 71, body weight was 96.3% of the control group value. A significant increase (p≤ 0.01) in body weight gain occurred on study days 36 to 43. This increase was not considered related to the test item because it did not persist.
Gestation body weights were significantly reduced (p≤ 0.05 to p≤ 0.01) for gestation days 7, 10 and 14 compared to the concurrent control group values. Body weight gains were significantly reduced (p≤ 0.05) for gestation days 0 to 7. Lactation body weights were reduced or significantly reduced (p≤ 0.05 to p≤ 0.01) for all days of lactation evaluated from lactation days 0 to 56. Lactation body weight gains did not differ significantly from the control group values for any interval evaluated.

FOOD CONSUMPTION:
1) males
- 40 mg Mo/kg bw/day: average food consumption was not significantly different for study days 1 to 71 and 1 to 143 compared to the control group value. Within this study, interval, significantly reduced (p≤ 0.05 to p≤ 0.01) food consumption occurred for 22 to 29, 29 to 36, 36 to 43, 106 to 113, 113 to 120, 120 to 122, 129 to 136 and 136 to 143. These statistically significant differences were not considered related to the test substance because these differences were transient.

2) females
- 40 mg Mo/kg bw/day: average food consumption was comparable between dose group and the control group and with no statistically significant differences occurring for study days 1 to 71, gestation days 0 to 20 and lactation days 0 to 14 and for any interval evaluated compared to the control group value for the premating, gestation and lactation periods.

COMPOUND INTAKE (via diet)
1) males
40 mg Mo kg bw/day:
- lowest average daily consumed molybdenum dose: 35.3 mg Mo kg bw/day
- highest average daily consumed molybdenum dose: 46.8 mg Mo kg bw/day
Average daily consumed dose molybdenum dose: 41.3 mg Mo kg bw/day

2) females
40 mg Mo kg bw/day (precohabitation):
- lowest average daily consumed molybdenum dose: 37.2 mg Mo kg bw/day
- highest average daily consumed molybdenum dose: 44.9 mg Mo kg bw/day
Average daily consumed dose molybdenum dose: 40.8 mg Mo kg bw/day

40 mg Mo kg bw/day (gestation):
- lowest average daily consumed molybdenum dose: 42.2 mg Mo kg bw/day
- highest average daily consumed molybdenum dose: 47.2 mg Mo kg bw/day
Average daily consmed dose molybdenum dose: 44.7 mg Mo kg bw/day

40 mg Mo kg bw/day (lactation):
- lowest average daily consumed molybdenum dose: 41.4 mg Mo kg bw/day
- highest average daily consumed molybdenum dose: 59.9 mg Mo kg bw/day
Average daily consmed dose molybdenum dose: 49.4 mg Mo kg bw/day

WATER CONSUMPTION
1) males
- 40 mg Mo/kg bw/day: average water consumption was slightly reduced (93.9% to 96.1%) between the dose group and the control group value for study days 1 to 71 and 1 to 143. Average water consumption was significantly increased (p≤ 0.05) for study days 1 to 8 in the dose groups compared to the control group value. Within this study interval, reduced or significantly reduced (p≤ 0.05) water consumption occurred in the dose group throughout the dosing interval.

2) females
- 40 mg Mo/kg bw/day: average water consumption was slightly reduced compared to the control group value for study days 8 to 15 through study days 64 to 71. Average water consumption was 94.9% of the control group value for study days 1 to 71, 93.2% for gestation days 0 to 20 and 96.4% for lactation days 0 to 14.

ORGAN WEIGHT FINDINGS INCLUDING ORGAN / BODY WEIGHT RATIOS
There were no adverse effects of treatment on organ weights or organ/body weight ratios in males or females
1) males
- 40 mg Mo/kg bw/day: terminal body weight was significantly reduced (p≤ 0.01,- 9.2%) compared to the control group value. The absolute weight of the right epididymis and liver were significantly reduced (p≤ 0.01) compared to the control group value. Furthermore, the ratio of the weight of brain and thyroid/parathyroid to the terminal body weight were both significant increased (p≤ 0.05 to p≤ 0.01) compared to the control group values. The ratio of the weight of the liver to the terminal body weight was significantly reduced (p≤ 0.01) compared to the control group values.

The other changes in organ weights (epididymis) or ratio of the organ weight (brain, thyroid/parathyroid) to the terminal body weight were not considered related to the test item because there was no adverse histopathology in these organs. In addition none of these differences were considered adverse because the differences were less than 10% of the control group values with the exception of the ratio of the thyroid/parathyroid to the terminal body weight, which was greater than 10% but the absolute weights did not differ among the groups.

2) females
- 0 and 40 mg Mo/kg bw/day: terminal body weight was comparable among the groups.
- 40 mg Mo/kg bw/day: ratio of the spleen weight to the terminal body weight was significantly increased (p≤ 0.05) compared to the control group value. The difference in the ratio of the organ weight to the terminal body weight was not considered adverse because no histopathological changes occurred in these organs. The increase in the ratio of the spleen weight to the terminal body weight was within 12.12% of the control group value.

GROSS PATHOLOGICAL FINDINGS
1) males and females
- 40 mg Mo/kg bw/day: no gross lesions related to sodium molybdate dihydrate at a dose of 40 mg Mo/kg bw/day diet occurred.

- 0 mg Mo/kg bw/day: one male had a small epididymides and small and flaccid testes. One non-pregnant female had a uterus that was diagnosed with pyometra with 15 to 20 mL of cloudy fluid (normal finding for a cycling non-pregnant rat). Another female had a small thymus.
- 40 mg Mo/kg bw/day: one male had a kidney with two clear fluid-filled cysts as well as a larger spleen and a large red liver with numerous clear fluid-filled cysts. One non-pregnant female had a uterus that was diagnosed with pyometra with 15 to 20 mL of cloudy fluid (normal finding for a cycling non-pregnant rat). Another female had a fractured palate.

HISTOPATHOLOGICAL FINDINGS - NON-NEOPLASTIC
- 40 mg Mo/kg bw/day: no test item-related microscopic findings were noted.

- 40 mg Mo/kg bw/day: a statistically significant increase was noted in the number of primordial follicles in left ovaries compared to the control group. Since there was no significance in the right ovaries or total count for both left and right ovaries, and the slight increase observed in treatment group versus the control group was not indicative of a clear pattern or trend, the changes observed were most likely due to natural variability among animals, and not to administration of the test item in the adult female rats.
The increased primordial follicles counts were not considered toxicologically important or adverse because: 1) there was no effect on fertility in either generation; 2) there was no treatment related observations on the histologic evaluation of the ovaries from the high dose group animals i.e., orderly maturation of the primary follicles to the subsequent stages, 3) the values were all within the historical control range and 4) no effect on ovarian weight or the ratio of these weights to the terminal body weight.

REPRODUCTIVE FUNCTION: OESTRUS CYCLE
- 40 mg Mo/kg bw/day: exposure did not affect premating oestrous cycling.

REPRODUCTIVE FUNCTION: SPERM MEASURES
- 40 mg Mo/kg bw/day: exposure to sodium molybdate dihydrate of 40 mg Mo/kg bw/day in diet did not affect sperm motility, concentration or morphology in the parental generation.

- 40 mg Mo/kg bw/day: percent of sperm with no head was significantly increased (p≤ 0.05) compared to the control value. This increase was not considered adverse because was within the historical control range for the laboratory (historical control data: detached head was 6.1% with a range of 1.0% to 19.4%). Although a slight increase (not statistically significant) was observed in the percent of no head sperm. These increases were not considered treatment-related because (1) in both cases, they were largely attributable to one male each with a high incidences of abnormal sperm (2) no evidence of an increase in the percent sperm with no head was observed among F1 males given up to 40 mg Mo/kg bw/day in the diet or drinking water, and (3) the percent sperm with no head was within the historical control range. There was no clear evidence of any compound-related effect on sperm concentration, spermatid concentration, sperm motility or morphology at dose levels up to 40 mg Mo/kg bw/day by either route of administration in either generation.
Apparent differences in sperm density and motility, and spermatid density between the two generations (including in the controls) was due to an upgrade in the CASA software. After revalidation, the gating parameters used for the second generation differed from the first generation resulting in lower density measurements and higher motility counts in the first generation and higher density counts with lower percentage motility in the second generation. The gating affects what the software will identify as a sperm vs. debris, and with more debris identified as sperm, the motility will appear lower.


REPRODUCTIVE PERFORMANCE
- 40 mg Mo/kg bw/day: exposure to test item at 40 mg Mo/kg bw/day in the diet did not affect mating in the male or female rats as all rats mated. . Pregnancy resulted in 19/24 (79.2%) rats (pregnancy percentage was not considered adverse, because: pregnancy rates of approx. 80% or higher are generally considered normal for the laboratory; pregnancy rate in the F1 generation was over 90%, demonstrating normal variation in these groups). All rats mated within approx. 2.7 days of being placed into cohabitation.
All other mating and fertility parameters (rats with confirmed mating dates during the first week of cohabitation and rats inseminated per rats in cohabitation) were comparable among the groups.

- 40 mg Mo/kg bw/day: natural delivery observations were unaffected by dosages of 40 mg Mo/kg bw/day. Values for the durations of gestation, averages for implantation sites per delivered litter, the gestation index and of dams with all pups dying were comparable among the control and diet group and did not significantly differ.

Key result

Dose descriptor:

LOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Remarks:

equivalent to 40 mg Mo/kg bw/day

Sex:

male/female

Basis for effect level:

body weight and weight gain

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1) males - body weights were reduced or significantly reduced (p≤ 0.05 to p≤ 0.01) compared to the concurrent control group for each weekly interval from Days 22 to 106 postpartum (last weight prior to cohabitation). On Day 106 postpartum, body weight was 92.9% of the control group value. Body weights continued to be significantly reduced (p≤ 0.05 to p≤ 0.01) post cohabitation until euthanasia. On study day 162 body weight was 92.4% of the control group value.
Body weight gains compared to the concurrent control group were reduced and/or significantly reduced (p≤ 0.05 to p≤ 0.01) for Days 36 to 43, 43 to 50, 64 to 71, 22 to 106 and 22 to 162 postpartum

2) Females - body weights and body weight gains were comparable among the groups for the pre-mating period. Day 106 postpartum, body weight was 94.5% of the control group value. Body weights for Days 85 and 99 postpartum and body weight gains for Days 22 to 106 postpartum were significantly reduced (p≤ 0.05) Days 78 to 85 postpartum.
Gestation body weights were significantly reduced (p≤ 0.01) for gestation days 0, 14 and 20. Lactation body weights were significantly reduced (p≤ 0.05 to p≤ 0.01) for all days of lactation evaluated from lactation days 0 to 21, except on lactation day 14. Lactation body weight gains did not differ significantly except for lactation days 14 to 21 where a significant body weight loss occurred (p≤ 0.01).

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Details on results:

NOTE: Results of the F1 generation (postweaning though pairing to termination) are reported in this section, since these animals were considered to be the second parental generation (P1).
Effects that are considered as adverse and/or treatment related are described in the individual fields above.
Effects that are considered as non-adverse and/or not treatment-related are described in this field "Details on results".

CLINICAL SIGNS
1) males
- 40 mg Mo/kg bw/day: all clinical observations were considered unrelated to the test substance (observations occurred in only one or two rats; and/or observation is common in this species and strain), which included: ungroomed coat, soft or liquid feces, incisors missing/broken and swollen ears.

2) females
- 40 mg Mo/ kg bw/day: no adverse clinical observations related to the test substance occurred during the premating, gestation or lactation periods.

- all clinical observations were considered unrelated to the test substance (observations occurred in only one or two rats; and/or observation is common in this species and strain), which included: vocalization to touch, sparse hair coat and post parturition vaginal bleeding.

MORTALITY
- 40 mg Mo/kg bw/day: no unscheduled deaths occurred in the F1 generation.

The only early death occurred in the control gruop, but this was clearly not related to the test item:
- 0 mg Mo/kg bw/day: one male rat was euthanized on Day 158 postpartum due to adverse clinical observations. This rat had limited use of the hindlimbs during the 23rd week postpartum. The rat was gaining weight until Day 155 postpartum. Food and water consumption was comparable to other rats in the group. Necropsy revealed a flaccid testis. At necropsy, there was an abnormal appearance of the right testes that correlated with marked degeneration of the seminiferous tubules, microscopically. Also seen were mild hypospermatogenesis and minimal cellular debris in the epididymis and minimal nephropathy in the kidney. The cause of the acute hindlimb paralysis could not be determined by the gross and microscopic findings which were considered incidental.

BODY WEIGHT AND WEIGHT CHANGES
1) males
- 40 mg Mo/kg bw/day: body weights were reduced or significantly reduced (p≤ 0.05 to p≤ 0.01) compared to the concurrent control group for each weekly interval from Days 22 to 106 postpartum (last weight prior to cohabitation). On Day 106 postpartum, body weight was 92.9% of the control group value. Body weights continued to be significantly reduced (p≤ 0.05 to p≤ 0.01) post cohabitation until euthanasia. On study day 162 body weight was 92.4% of the control group value.
Body weight gains compared to the concurrent control group were reduced and/or significantly reduced (p≤ 0.05 to p≤ 0.01) for Days 36 to 43, 43 to 50, 64 to 71, 22 to 106 and 22 to 162 postpartum.

2) females
- 40 mg Mo/kg bw/day: body weights and body weight gains were comparable among the groups for the pre-mating period. On Day 106 postpartum, body weight was 94.5% of the control group value. Body weights for Days 85 and 99 postpartum and body weight gains for Days 22 to 106 postpartum were significantly reduced (p≤ 0.05) compared to the control group value and also significantly reduced (p≤ 0.05) on Days 78 to 85 postpartum.
Gestation body weights were significantly reduced (p≤ 0.01) for gestation days 0, 14 and 20 compared to the concurrent control group values. Body weight gains did not differ significantly between the groups. Lactation body weights were significantly reduced (p≤ 0.05 to p≤ 0.01) for all days of lactation evaluated from lactation days 0 to 21, except on lactation day 14. Lactation body weight gains did not differ significantly from the control group except for lactation days 14 to 21 where a significant body weight loss occurred (p≤ 0.01).

FOOD CONSUMPTION
1) males
- 40 mg Mo/kg bw/day: average food consumption was significantly reduced (p≤ 0.05 to p≤ 0.01) for each weekly interval from Days 64 to 71 to 99 to 106 postpartum. Food consumption for Days 22 to 106 postpartum was 95.5% of the control group value. Post cohabitation significant reductions (p≤ 0.05 to p≤ 0.01) in food consumption occurred on Days 127 to 134 and 141 to 148 postpartum. Food consumption for Days 22 to 162 postpartum was 94.9% of the control group value.

2) females
- 40 mg Mo/kg bw/day: average food consumption was comparable between the treatment group and controls with no statistically significant differences occurring for study days 1 to 71, gestation days 0 to 20 and lactation days 0 to 14 and for any interval evaluated compared to the control group value except for a significantly increased (p≤ 0.05) average food consumption for Day 22 to 29 postpartum and transient reductions (p≤ 0.01) during gestation for gestation days 10 to 14 and 18 to 20.

COMPOUND INTAKE (via diet)
1) males
40 mg Mo/kg bw/day:
- lowest average daily consumed molybdenum dose: 19.4 mg Mo kg bw/day
- highest average daily consumed molybdenum dose: 59.3 mg Mo kg bw/day
Average daily consmed dose molybdenum dose: 41.3 mg Mo kg bw/day

2) females
40 mg Mo/kg bw/day (precohabitation):
- lowest average daily consumed molybdenum dose: 25.4 mg Mo kg bw/day
- highest average daily consumed molybdenum dose: 54.4 mg Mo kg bw/day
Average daily consmed dose molybdenum dose: 42.6 mg Mo kg bw/day

40 mg Mo/kg bw/day (gestation):
- lowest average daily consumed molybdenum dose: 36.0 mg Mo kg bw/day
- highest average daily consumed molybdenum dose: 46.2 mg Mo kg bw/day
Average daily consmed dose molybdenum dose: 40.5 mg Mo kg bw/day

40 mg Mo/kg bw/day (lactation):
- lowest average daily consumed molybdenum dose: 33.6 mg Mo kg bw/day
- highest average daily consumed molybdenum dose: 48.0 mg Mo kg bw/day
Average daily consmed dose molybdenum dose: 40.7 mg Mo kg bw/day

WATER CONSUMPTION
1) males
- 40 mg Mo/kg bw/day: average water consumption was generally comparable between the control and treatment group. Average water consumption for Days 22 to 106 postpartum was 102% compared to the control group value and 101.3% of the control group value for Days 22 to 162 postpartum.

2) females
- 40 mg Mo/kg bw/day: average water consumption was comparable between the control group and treatment group. Average water consumption was 97.7% of the control group value for Days 22 to 106 postpartum and 91.1% of the control group value for gestation days 0 to 20 and 92.8% of the control group value for lactation days 0 to 14. During the gestation period, average water consumption was significantly reduced (p≤ 0.05) for gestation days 10 to 14.

ORGAN WEIGHT FINDINGS INCLUDING ORGAN / BODY WEIGHT RATIOS
1) males
- 40 mg Mo/kg bw/day: terminal body weight was significantly reduced (p≤ 0.05, -7.7%) compared to the control group value. The absolute weight of the liver was significantly reduced (p≤ 0.01) compared to the control group value. The reduction reflected the lower body weight in this group and was not considered adverse as no histopathology related to the test item occurred. The ratio of the weight of the thyroid/parathyroid to the terminal body weight was significantly increased (p≤ 0.05) compared to the control group values. This was not considered adverse as no histopathology related to the test item occurred.

2) females
- 40 mg Mo/kg bw/day: a significant reduction (p≤ 0.05) in the terminal body weight compared to the control group values was observed.
The absolute weights of the pituitary, brain, liver, kidneys, adrenals, spleen, thymus, ovaries, uterus and thyroid/parthyroid and the ratio of these organ weights to the terminal body weights were not affected by exposure in diet at a dose of 40 mg Mo/kg bw/day.

GROSS PATHOLOGICAL FINDINGS
- 5, 17 and 40 mg Mo/kg bw/day: no gross lesions related to sodium molybdate dihydrate at doses as high as 40 mg Mo/kg bw/day diet occurred.

None of the gross lesions observed were considered related to the test item. The renal pelvic dilataion lesion was observed in control and treated animals, the incidence was not dose-dependent and there was no histopathological changes related to the test item in these organs in the high dose diet group and the lesion is known to occur spontaneously.
The following findings were recorded:
1) males
- 0 mg Mo/kg bw/day: one male rat had a flaccid testis. Another male rat had numerous red areas on the thymus and a firm yellow mass on the testis that when cut revealed a yellow caseous material. This male did not sire a litter.
- 40 mg Mo/kg bw/day: one male rat was euthanized on Day 24 postpartum and was replaced owing to adverse clinical signs including bradypnea and severe dehydration. This rat had moderate dilatation of each renal pelvis and large kidneys. Another two male rats had slight to moderate dilation of the renal pelvis. Lastly, two male rats had a red area on the thymus.

2) females
- 0 mg Mo/kg bw/day: one female rat had red pinpoint areas on the thymus. This rat was never pregnant.
- 40 mg Mo/kg bw/day: one female rat had bilateral dark red adrenal glands. This rat was never pregnant and did not mate

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC
- 40 mg Mo/kg bw/day: no test item-related microscopic findings were noted.

- corpora lutea were present for all scheduled euthanasia animals evaluated in all groups in the F1 Generation.
- ovarian follicle counts demonstrated normal variability in this strain of rat.
- values in the treated group were higher than the control group (not considered toxicologically important or adverse, because: no effect on fertility in either generation; no treatment related observations on the histologic evaluation of the ovaries i.e., orderly maturation of the primary follicles to the subsequent stages; values were all within the historical control range and no effect on ovarian weight or the ratio of these weights to the terminal body weight.)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

NOTE: results of the F1 generation (postweaning through pairing until termination) are reported in this section, since these animals were considered to be the second parental generation (P1).
Effects that are considered as adverse and/or treatment related are described in the individual fields above.
Effects that are considered as non-adverse and/or not treatment-related are described in this field "Details on results".

REPRODUCTIVE FUNCTION: ESTROUS CYCLE
- 40 mg Mo/kg bw/day: exposure to sodium molybdate dihydrate at doses of molybdenum of 40 mg Mo/kg bw/day in the diet did not affect premating estrous cycling.

REPRODUCTION FUNCTION: SPERM MEASURES
- 40 mg Mo/kg bw/day: exposure to sodium molybdate dihydrate of 40 mg Mo/kg bw/day in diet did not affect sperm motility, concentration or morphology. The percent of sperm with no head was not significantly different from the control value.

REPRODUCTIVE PERFORMANCE
- 40 mg Mo/kg bw/day: exposure to sodium molybdate dihydrate at 40 mg Mo/kg bw/day in the diet did not affect mating in the male or female rats. Of the 23/24 (95.8%) rats mating, pregnancy resulted in 22/24 (91.7%) rats. All rats mated within approximately 3.3 days of being placed into cohabitation.
All other mating and fertility parameters (rats with confirmed mating dates during the first week of cohabitation and rats inseminated per rats in cohabitation) were comparable among the groups.

Natural delivery observations were unaffected by doses of 40 mg Mo/kg bw/day. Values for the durations of gestation, averages for implantation sites per delivered litter, the gestation index, and of dams with stillborn pups or total litter loss were comparable between the control and diet and did not significantly differ.

Key result

Dose descriptor:

LOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Remarks:

equivalent to 40 mg Mo/kg bw/day

Sex:

male/female

Basis for effect level:

body weight and weight gain

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Effects that are considered as adverse and/or treatment related are described in the individual fields above.
Effects that are considered as non-adverse and/or not treatment-related are described in this field "Details on results".

CLINICAL SIGNS:
- 40 mg Mo/kg bw/day: no adverse clinical observations occurred in the pups at a dose of the test substance of 40 mg Mo/kg bw/day.

No clinical observations considered related to the test item occurred as none occurred in more than one to three litters and/or the observation was not dose dependent. These observations included: cold to touch, a scab on the head, neck and/or base of tail, dehydration, or tail not present (presumed cannibalized).

- 40 mg Mo/kg bw/day: treatment did not affect the onset or development of eye opening, hair growth, incisor eruption or pinna unfolding. No statistically significant differences occurred among the groups for any parameter evaluated.

MORTALITY / VIABILITY
- 0 and 40 mg Mo/kg bw/day: viability and lactation indices, and live litter size at weighing were comparable among the control and diet group and did not significantly differ.

BODY WEIGHTS AND WEIGHT CHANGES
- 0 and 40 mg Mo/kg bw/day: values for the pup weights were comparable among the control and diet group and did not significantly differ.

GROSS PATHOLOGICAL FINDINGS
- 40 mg Mo/kg bw/day: no adverse necropsy observations occurred in the pups at a dose of the test substance of 40 mg Mo/kg bw/day.

No necropsy observations were considered related to the test item as none occurred in more than one to three litters
All pups necropsied on postnatal day 21 or 23 appeared normal.

HISTOPATHOLOGICAL FINDINGS
- 40 mg Mo/kg bw/day: no test item-related microscopic findings were noted.

SEXUAL MATURATION
1) males
- 5, 17, and 40 mg Mo/kg bw/day: preputial separation and the average body weight of the male rats on the day of preputial separation were not affected by exposure to the test item at a dose of molybdenum of 40 mg Mo/kg bw/day in the diet.

2) females
- 5, 17, and 40 mg Mo/kg bw/day: vaginal patency and the average body weight of the female rats on the day of vaginal patency were not affected by exposure to the test item at a dose of molybdenum of 40 mg Mo/kg bw/day in the diet.

ORGAN WEIGHTS INCLUDING ORGAN / BODY WEIGHT RATIONS
- 40 mg Mo/kg bw/day: no effect on organ weights (brain, spleen, and thymus) or ratio of the organ weight to the terminal body weight occurred in the pups at a dose of the test substance of 40 mg Mo/kg bw/day.

Key result

Dose descriptor:

NOAEL

Generation:

F1

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

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Effects that are considered as adverse and/or treatment related are described in the individual fields above.
Effects that are considered as non-adverse and/or not treatment-related are described in this field "Details on results".

CLINICAL SIGNS
- 40 mg Mo/kg bw/day: no adverse clinical observations occurred in the pups.

- 40 mg Mo/kg bw/day: maternal dose of 40 mg Mo/kg bw/day did not affect the onset or development of eye opening, hair growth, incisor eruption or pinna unfolding.

MORTALITY / VIABILITY
- 0 and 40 mg Mo/kg bw/day: viability and lactation indices, and live litter size at weighing were comparable among the control and diet group and did not significantly differ.

BODY WEIGHTS AND WEIGHT CHANGES
- 0 and 40 mg Mo/kg bw/day: values for the the pup weights were comparable among the control and diet group and did not significantly differ.

SEXUAL MATURATION
- 40 mg Mo/kg bw/day: no adverse effect was observed

ORGAN WEIGHT FINDINGS INCLUDING ORGAN / BODY WEIGHT RATIOS
- 40 mg Mo/kg bw/day: no test substance-related effects on organ weights occurred in the pups.

- 40 mg Mo/kg bw/day: there was a statistically significant (p≤ 0.01) reduction in the thyroid/parathyroid weight in the females in comparison with the control group value (not considered to be test substance related).

GROSS PATHOLOGICAL FINDINGS
- 40 mg Mo/kg bw/day: no adverse necropsy observations occurred in the pups.

All pups necropsied on postnatal day 21 through 24 appeared normal except for 2 pups from different litters in the 0 Mo/kg bw/day group. The pups in the control group had clear fluid filled cysts in the kidney.

Necropsy of found dead and stillborn pups revealed 5 pups from 3 litters in the 40 mg Mo/kg bw/day diet group had no milk in their stomach compared to 2 pups from 2 litters in the control group . All other found dead or stillborn pups appeared normal.

HISTOPATHOLOGICAL FINDINGS - NON-NEOPLASTIC
- 40 mg Mo/kg bw/day: no test item-related microscopic findings were noted.

- all the males and females were, as expected, sexually immature on postnatal day 22. In the males there was no active spermatogenesis in the testes and in the females no corpora lutea present in the ovaries in control or treated groups.

Key result

Dose descriptor:

NOAEL

Generation:

F2

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

Reproductive effects observed:

no

Conclusions:

Introductory remark:
The main driver for conducting this 2-generation study was for regulatory purposes in the United States of America. In the past, regulations deriving health thresholds for molybdenum have often relied on the non-guideline study by Fungwe et al. (1990) in which rats were exposed to molybdenum via drinking water, and US EPA were seeking a more robust study. Therefore, this 2-generation study was also conducted via drinking water, to see if it would be possible to replicate the findings reported by Fungwe et al. (1990). The range-finder and the main study each included three dose levels via drinking water. Since for humans the exposure via the diet is the more relevant route of exposure, the range-finder also included 3 dose levels via the diet. For the main study, only the top-dose was administered via the diet. The US EPA Office of Water was involved in the decision to select drinking water as the main route of administration in the main study.
For technical reasons, four separate study records had to be prepared in IUCLID for the drinking water and diet parts of the range-finder and main study, respectively. All parts of the study should be considered together.
This study record is for the main study, dietary administration part of the study.
Summary:
The effects of sodium molybdate dihydrate on the male and female reproductive systems in Crl:CD (SD) Sprague Dawley rats, including gonadal function, oestrous cycles, mating behaviour, conception, gestation, parturition, lactation, weaning and the growth and development of offspring were investigated in an OECD Test Guideline 416 study. The dose levels were selected based on a previous sub-chronic (90 day) study, on a developmental toxicity study and on a rangefinder study in rats in which clear effects were observed on bodyweight at the top dose level of 40 mg Mo/kg bw/day. In the sub-chronic study, the top dose level of 60 mg Mo/kg bw/day produced excessive bodyweight loss in males, and renal toxicity in females. The dose levels were 5,17 and 40 mg Mo/kg bw/day in drinking water and 40 mg Mo/kg bw/day.
The male rats were directly exposed to the test or control diet or drinking water for at least 10 weeks before cohabitation, during the cohabitation, and continuing through to the day of euthanasia. The female rats were directly exposed to the test or control diet or drinking water for at least 10 weeks before cohabitation, during the cohabitation, gestation, littering and post-partum periods (lactation period; DL) and continuing through to the day of euthanasia.
F1 generation pups were directly exposed to the test and/or carrier control substances after they begin consuming food/water (approximately on Day 15 postpartum). Pups will have been exposed to the test and/or carrier control substances during maternal gestation (in utero exposure) or via maternal milk during the lactation period. The selected F1 generation weaned pups were directly exposed to the test or control diet or drinking water at least 10 weeks before cohabitation (beginning on Day 22 postpartum), during the cohabitation, gestation, littering and post-partum periods (lactation period; DL) and continuing through to the day of euthanasia.
F2 generation pups were directly exposed to the test and/or carrier control substances after they begin consuming food/water (approximately on Day 15 postpartum). Pups will have been exposed to the test and/or carrier control substances during maternal gestation (in utero exposure) or via maternal milk during the lactation period.

Results:
All diet and water formulations that were analyzed were found to be within or very close to the planned quality control /quantity assurance parameters.
No deaths related to sodium molybdate dihydrate occurred in either the P or F1 generations. One P generation 40 mg Mo/kg bw/day drinking water group male rat was found dead on Study Day 62 (DS 62) which appeared to be secondary to aspiration of bedding material. In the P generation, one control group female rat was euthanised on Day 23 of lactation, and one 40 mg Mo/kg bw/day diet dose group female rat was euthanized on Day 43 of lactation due to adverse clinical observations (swollen limb, fractured palate) that appeared to be due to accidental injuries not related to sodium molybdate dihydrate.
One F1 generation control group male rat was euthanized on Day 158 postpartum and one F1 generation male rat in the 17 mg Mo/kg bw/day drinking water group was euthanized on Day 44 postpartum due to adverse clinical observations. One F1 generation female 17 mg Mo/kg bw/day drinking water dose group rat died during blood collection on Day 30 of lactation. All other weaned F1 generation rats survived to scheduled euthanasia.

Groups Exposed to the Test Material in the Diet (40 mg Mo/kg bw/day):
P Generation Male and Female Rats and F1 Generation to Weaning
Note in the IUCLID report results for the P Generation are recorded as P0 Generation
No adverse clinical observations related to the test substance occurred male rats or female rats during the premating, gestation or lactation periods. Five male rats in the group given 40 mg Mo/kg bw/day in the diet had vocalization to touch. Any toxicological significance of this is unclear. .
Body weights and body weight gains in male rats were reduced or significantly reduced in the 40 mg Mo/kg bw/day. The average body on study day 71 (last weight prior to cohabitation) weight was 94.1% o and on study day 143 body weight was 91.4% of the control group.
Body weights and body weight gains in female rats were not significantly decreased during the pre-mating period. However on DS 71, the average body body weight was 96.3% of the control group. Gestation body weights were significantly reduced by 5.4 g to 6.9 g for gestation days 7, 10 and 14 compared to the control group values. Body weight gains were significantly reduced for DGs 0 to 7. Lactation average body weights but not body weight gains were significantly reduced for all days of lactation evaluated
The absolute weight of the right epididymis and absolute and relative weight of the liver was significantly reduced compared to the control group values. The ratio of the weight of the thyroid/parathyroid and brain to the terminal body weight in the 40 mg Mo/kg bw/day diet dose group was significantly increased compared to the control group value. These changes in organ weight and ratio to the terminal body were not considered adverse as no histopathological changes occurred in these tissues.


No effects were observed on food or water consumption. No effects were observed on premating oestrous cycling or mating or fertility, sperm motility, concentration or morphology . Natural delivery was unaffected.
F1 Generation Male and Female Rats and F2 Generation to Weaning
No adverse clinical were observation during the premating, gestation or lactation periods.
Body weights in the F1 male rats were significantly reduced for each weekly interval from study days 78 to 106 (92.9% ) postpartum (last weight prior to cohabitation) and to study day 162 (92.4%). Body weight gains in the F1 male rats were also were significantly reduced for study days 22 to 106 and 22 to 162 postpartum.
Body weights in the F1 female rats were not significantly different from the control body weights during the pre-mating period being 94.5% of control on study day 106 postpartum Body weight gain for study days 22 to 106 postpartum was significantly reduced (93.6%). Gestation body weights were significantly reduced on gestation days 0, 14 and 20 (92.1% of gestation day 20). Lactation body weights were significantly reduced for all days of lactation evaluated from lactation day 0 to 21 (90.6% on lactation day 21)

Terminal body weight in F1 male rats was significantly reduced (92.3%). The absolute weight of the liver, but not the relative liver weight was significantly reduced to 86.2% of controls. The ratio of the weight of the thyroid/parathyroid to the terminal body weight by 12% compared to the control group value. These differences were not considered adverse because there were no histopathological changes in these organs.
Terminal body weight in F1 female rats was significantly reduced to 94.4% but no effects on the organ weight or ratio of the organ weight to the terminal body weight were observed.
The average food consumption in F1 male rats was significantly reduced for each weekly interval from study days 64 to 71 and 99 to 106 postpartum with overall consumption on study days 22-162 being 94.9% of control group.
No no statistically significant differences in average food consumption in F1 female rats was observed
Average water consumption in F1 male and female rats was generally comparable between the control, but during the gestation period, average water consumption was significantly reduced compared with controls on DGs 10 to 14. No adverse effects were observed on preputial separation, vaginal patency and the average body weight on the day of sexual maturation.
In F1 rats, no effects were observed on premating estrous cycling or mating. Of the 24 pairs of rats mating, pregnancy occurred in 22 females (91.7%). No effects were observed on sperm motility, concentration or morphology. No effect was observed on natural delivery.
In F2 pups no adverse clinical or necropsy effect were observed. No effects were observed on on organ weights with the exception of thyroid/parathyroid which was not considered adverse because there were no histopathological changes in these organs

Conclusion based on both Drinking Water and Diet Dosed Groups

Based on the results of this study the no-observed-adverse-effect level (NOAEL) for paternal and maternal toxicity was 17 mg Mo/kg bw/day based on reductions in body weight and food consumption in the 40 mg Mo/kg bw/day dose groups (diet). The NOAEL for males and females of the P and F1 generations for reproductive toxicity, including mating and fertility and the growth and development of the F1 generations to adulthood and the growth and development up to weaning of the F2 generation, was 40 mg Mo/kg bw/day whether exposure occurred in the diet or drinking water.

The top dose level of 40 mg Mo/kg bw/day is approximately 20,000 times greater than the normal total dietary and water intake of molybdenum of 2 µg Mo/kg bw/day in humans (general population).

Executive summary:

In an OECD Test Guideline 416 multigenerational study, groups of 24 male and 24 female Sprague-Dawley ratswere administered sodium molybdate dihydrate at 0, 5, 17, or 40 mg molybdenum (Mo)/ kg bw/day in the drinking water and 40 mg molybdenum (Mo)/ kg bw/day in diet over two generations to assess reproductive toxicity. No adverse effect on reproductive function was observed at any dose level in either generation as indicated by no significant dose-related effect on oestrus cycles, sperm parameters, mating, fertility, gestation, litter size, pupsurvival, growth or postnatal development. Serum levels of molybdenum were increased in a dose-related manner. The No Observed Adverse Effect Levels (NOAEL) are 17 mg Mo/kg bw/day for systemic toxicity and 40 mg Mo/kg bw/day for reproductive toxicity. The drinking water administration results are given in a separate entry.

Reference 2

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-07-07 to 2017-03-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

2001-01-22

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: controlled room temperature

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The Sprague Dawley rat was chosen as the animal model for this study because: 1) it is an accepted rodent species for preclinical toxicity testing by regulatory agencies; 2) this species and strain has been demonstrated to be sensitive to developmental toxicants; and 3) historical data and experience exist at the testing facility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC, United States
- Age upon arrival at laboratory: males: approx. 45 days; females : approx. 37 days
- Weight upon arrival at laboratory: males: 150 - 196 g; females: 77 - 108 g
- Weight at randomization and study assignment: males: 174 - 211 g; females: 91 - 120 g
- Housing: individually housed in solid-bottomed cages, except during the cohabitation and postpartum periods and during urine collection; during cohabitation: each pair of rats was housed in the male rat’s nesting box; during the postpartum period: each dam and delivered litter were housed in individual nesting boxes until weaning; duration of urine collection: rats selected for collection were housed in a stainless steel, wire bottom metabolism cage; Nesting material (Bed-o'Cobs®) was provided.
- Diet (ad libitum): Certified Rodent Diet® #5002 (PMI® Nutrition International)
- Water (ad libitum): during the acclimation period: water was passed through a reverse osmosis membrane and chlorine was added; during the exposure period: R.O. deionized water without chlorine
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19°C to 25°C)
- Relative humidity: 30% to 70%
- Air changes (per hr): ≥ 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: drinking water

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF TEST SUBSTANCE
- test substance dosing formulations were prepared based on sponsor instructions and a previous range-finding study (please refer to study record on the range-finding study in the same IUCLID section 7.8.1) at appropriate concentrations to meet dose level requirements.
- the formulations prepared in reverse osmosis membrane-processed deionized water were prepared at least every 14 days and stored at room temperature until use.
- a concentration of the test and/or control substance in the water was offered to the rats, and the mg/kg/day doses consumed were calculated and presented for periods corresponding to body weight and food consumption observations

Details on mating procedure:

Parental (P) generation
- M/F ratio per cage: 1 male / 1 female
- Length of cohabitation: maximum of 10 days
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as gestation day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individual housing
- Female rats not mated after completion of the 10-day cohabitation period were considered to be at gestation day 0 on the last day of cohabitation.

F1 generation
- M/F ratio per cage: 1 male / 1 female
- Length of cohabitation: maximum of 17 days
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as gestation day 0 of pregnancy
- After 10 days of unsuccessful pairing replacement of first male by another male and remained in cohabitation for a maximum of 7 additional days.
- After successful mating each pregnant female was caged (how): individual housing
- Female rats not mated after completion of the 17-day cohabitation period were considered to be at gestation day 0 on the last day of cohabitation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for analysis of concentration of molybdenum according to the following schedule:
- first preparation
- at approximately monthly intervals
- last preparation
Analyses described below were performed by inductively coupled plasma - mass spectroscopy (ICP-MS).

Analysis of concentration of molybdenum:
- duplicate middle sets of samples for each sampling time point were collected
- concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% of theoretical concentration.
- each individual sample concentration result was considered acceptable if it was within or equal to ± 15%.

Analysis of stability:
Stability analyses performed previously in a dose range finder study (please refer to section 7.8.1 Toxicity to reproduction: s_Hoberman_2016_dose-range finder (drinking water)) demonstrated that the test substance is stable in the drinking water when prepared and stored under the same conditions at concentrations bracketing those used in the present study (did not cover the low concentration of water at 13.1 ppm used in the current study).

Results:
- all water formulations that were analyzed were found to be within or very close to the planned quality control parameters set forth in the protocol, and thus appropriate for use.
- the amount of test substance used each week to prepare dosed water was adjusted weekly based on estimated average body weight for the next week of study, to achieve a daily exposure of 0, 5, 17 or 40 mg Mo/kg bw/day from the drinking water. This weekly adjusting of concentration was continued throughout the premating period for both males and females, and continued for the males until termination. During the gestation and lactation periods for the female rats, test water was adjusted weekly based on the day of gestation or lactation to provide test water with concentrations that would achieve the 0, 5, 17 or 40 mg Mo/kg bw/day doses from sodium molybdate dihydrate.
- homogeneity and stability of the dosed water for 4, 10 and 14 days room temperature and refrigerated were established during the conduct of dosage-range study (please refer to section 7.8.1 Toxicity to reproduction: s_Hoberman_2016_dose-range finder (drinking water)).

- concentration analyses: all analyzed samples were within or equal to the acceptance criteria of ± 10% for the drinking water (individual sample within or equal to ± 15% for the drinking water) of their theoretical concentrations (in exceptional cases values were higher than the target value, which was not considered to have an effect on the study).

Duration of treatment / exposure:

Parental (P) generation:
- males: at least 10 weeks before cohabitation, during the cohabitation, and continued through to the day before euthanasia
- females: at least 10 weeks before cohabitation, during the cohabitation, gestation, littering and lactation periods and continuing through to the day of euthanasia

F1 generation:
- pups were directly exposed after they begun consuming food/water (approx. on Day 14 postpartum).
- pups will have been exposed during maternal gestation (in utero exposure) or via maternal milk during the lactation period (Ref Karolina Kot et al 2019)
- the selected weaned pups were directly exposed at least 10 weeks before cohabitation, during the cohabitation, gestation, littering and lactation period and continuing through to the day of euthanasia.

F2 generation:
- pups were directly exposed after they begin consuming food/water (approx. on Day 14 postpartum).
pups will have been exposed during maternal gestation (in utero exposure) or via maternal milk during the lactation period (Ref Karolina Kot et al 2019)

Karolina Kot, et al, 2019, International Journal of Environmental Research and Public Health. Interactions between 14 Elements in the Human Placenta, Fetal Membrane and Umbilical Cord. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6540151/

Frequency of treatment:

ad libitum

Details on study schedule:

- Selection of parents from F1 generation when pups were 21 days of age (weaning day; at least one male pup and one female pup per litter were selected; selection of runts, or pups otherwise impaired, was avoided).

- F1 parental animals not mated until 10 weeks after selected from the F1 litters.

- Age at mating of the mated animals (F1 generation) in the study: approx. 90 days of age

Dose / conc.:

12.5 mg/kg bw/day (nominal)

Remarks:

equivalent to 5 mg Mo/kg bw/day

Dose / conc.:

42.5 mg/kg bw/day (nominal)

Remarks:

equivalent to 17 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:

24 males / 24 females

Control animals:

yes, concurrent no treatment

Details on study design:

The main driver for conducting this 2-generation study was for regulatory purposes in the United States of America. In the past, regulations deriving health thresholds for molybdenum have often relied on the non-guideline study by Fungwe et al. (1990) in which rats were exposed to molybdenum via drinking water, and US EPA were seeking a more robust study. Therefore, this 2-generation study was also conducted via drinking water, to see if it would be possible to replicate the findings reported by Fungwe et al. (1990). The range-finder and the main study each included three dose levels via drinking water. Since for humans the exposure via the diet is the more relevant route of exposure, the range-finder also included 3 dose levels via the diet. For the main study, only the top-dose was administered via the diet. The US EPA Office of Water was involved in the decision to select drinking water as the main route of administration in the main study.
For technical reasons, four separate study records had to be prepared in IUCLID for the drinking water and diet parts of the range-finder and main study, respectively. All parts of the study should be considered together.
This study record is for the main study, drinking water part of the study.
The dose levels were selected based on the results of previous sub-chronic and developmental toxicity studies conducted by the Sponsor, and a rangefinder study (please refer to separate study record in IUCLID section 7.8.1) in which clear effects were observed in the bodyweights at the top dose levels (40 mg Mo/kg bw/day). In the sub-chronic (90 day study) in rodents a top dose of 60 mg Mo/kg bw/day produced an excessive bodyweight loss in males (~15%), and renal toxicity in females (Murray et al., 2014)*. In the rangefinder study, doses as high as 40 Mo/kg bw/day were chosen and administered in the diet or water. The results of the rangefinder study are summarized below.
No deaths related to sodium molybdate dihydrate occurred in the rangefinder study (please refer to separate study record in IUCLID section 7.8.1). Administration of sodium molybdate dihydrate in the water or diet reduced body weight gains, body weights, water and feed consumption in the 40 mg Mo/kg bw/day exposure group. Within the intervals evaluated (premating, gestation, lactation) sporadic reductions in body weight, feed and water consumption occurred in the 3 and 20 mg Mo/kg bw/day exposure groups, and these reductions were not considered adverse.
Terminal body weights in the male rats were reduced in both the diet and water 40 mg Mo/kg bw/day exposure groups in the rangefinder. In the groups exposed via the diet, the absolute weight of the seminal vesicles with fluid was significantly reduced in the 20 and 40 mg Mo/kg bw/day dose groups compared to the control group value. The ratio of the weight of the left and right testes and the left cauda epididymis to the terminal body weight in the 40 mg Mo/kg bw/day diet dose group were all significant increased compared to the control group values.
The absolute weight of the seminal vesicles with fluid and the weight of the left kidney were significantly reduced in the 40 mg Mo/kg bw/day water dose groups compared to the control group value in the rangefinder. The ratio of the weight of the left and right testes to the terminal body weight in the 40 mg Mo/kg bw/day water dose group were both significantly increased compared to the control group values.
The changes in organ weights were not considered adverse in the rangefinder as there were no histological changes in any organ evaluated and the increased organ to body weight ratios indicated that the changes reflected the reduced terminal body weights.
Oestrous cycling, mating and fertility were not affected by exposures as high as 40 mg Mo/kg bw/day in the diet or water in the rangefinder. Sperm parameters were comparable among the diet and water exposure groups. A reduced number of females pregnant (6/10 pregnant) in the 40 mg Mo/kg bw/day water exposure group was observed. The litter size and survival to weaning of the F1 generation was not affected by maternal doses of sodium molybdate dihydrate in the diet or water.
Based on the results of this study doses as high as 40 mg Mo/kg bw/day were selected for the evaluation in the full multigenerational study since this resulted in a reduction in body weights in the rangefinder in excess of 10% in the males which is generally accepted as a maximum tolerated dose level. In addition, in females, in the diet administration study, body weights were significantly reduced to 90.1% of controls prior to cohabitation and also significantly reduced during gestation and lactation.
Exposure through the drinking water was used because of the apparent reduced number of females (6/10) in the 40 mg Mo/kg bw/day water exposure group in the rangefinder and in addition to assist in trying to replicate the findings reported by Fungwe et al. (1990) as agreed with the USA EPA (see first paragraph of this section) . A comparator group, at 40 mg Mo/kg bw/day the diet was also included in the study design to evaluate potential carrier differences.


*Reference:
- Murray FJ, Sullivan FM, Tiwary AK, Carey S. 90-Day subchronic toxicity study of sodium molybdate dihydrate in rats. Regulatory Toxicology and Pharmacology 2014; 70:579-588.

Positive control:

none

Parental animals: Observations and examinations:

1) PARENTAL (P) GENERATION / F1 GENERATION (producing F2 generation) Recorded as PARENTAL (P) GENERATION - P0 and F1 GENERATION (producing F2 generation) -P1

CAGE SIDE OBSERVATIONS: Yes
Time schedule:
- general health/ mortality and moribundity: twice daily, once in the morning and once in the afternoon.
- maternal observations: apparently pregnant females were observed frequently from the expected day of parturition to determine, where possible, onset and duration of parturition; thereafter, maternal observations were recorded at least once daily. Observed abnormal behaviour was recorded daily

DETAILED CLINICAL OBSERVATIONS: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily during exposure periods and prior to euthanasia
- during cohabitation, when two rats occupied the same nesting box with one water bottle (using the lower concentration available at the time, as applicable), replenishment of the water was documented
- water consumption was not tabulated after lactation day 14, when it was expected that pups would begin to consume water.

FURTHER OBSERVATIONS:
- urine collection: urine samples were collected approx. 24 hours on the following days:
P Generation (12 rats/group/sex):
males: study days 30, 31, 32 or 33 and on study days 106, 108, 109 or 110
females: study days 30, 31, 32 or 33, gestation day 30 or lactation days 25, 29, 30, 31, 32, 33, 34, 35 or 36
F1 Generation (12 rats/group/sex)
males: days 48, 49, 50 or 51 postpartum, and on days 144, 145 or 146 postpartum.
females: days 48, 49, 50 or 51 postpartum and gestation days 47or 48 or DL 23, 24, 25, 26, 27 or 28.
The urine samples were analysed for molybdenum and other elements.

-blood collection: blood was collected on the day before scheduled euthanasia (collection: morning and afternoon; 12 rats/group/sex (same animals selected urine collection)).
Serum samples were analysed for were analysed for molybdenum and other elements.

- mating performance: mating was evaluated daily during the cohabitation period and until confirmed by spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ.

- natural delivery observations: female rats were evaluated for adverse clinical signs (including any indication of prolonged parturition) and duration of gestation (gestation day 0 until the time the first pup was observed).

2) PARENTAL (P) GENERATION (P0)

CAGE SIDE OBSERVATIONS: Yes
Time schedule:
- general appearance: at least weekly during the acclimation period, on the day of randomization, daily during the dose period, and on the day of scheduled euthanasia

BODY WEIGHT: Yes
Time schedule for examinations:
- males: individually weighed on the day after arrival, at least weekly during the acclimation period, at least weekly during the dose period and on the day of scheduled euthanasia.
- females: individually weighed on the day after arrival, at least weekly during the acclimation period, at least weekly during the dose period on gestation days 0, 7, 10, 14, 20, 25, and weekly thereafter (as applicable) lactation days 0, 4, 7, 14, 21, 28, 35, 42, 49, 56 and 58 and the day of scheduled euthanasia

FOOD CONSUMPTION: Yes
- males: at least weekly during the dose period
- females: at least weekly during the dose period on gestation days 0, 7, 10, 14, 18, 20 and 25 and lactation days 0, 4, 7, 10, 14, 21, 28, 35, 42, 49, 56 and 58, and on the day of scheduled euthanasia.
- during cohabitation, when two rats occupied the same nesting box with one food jar (using the lower concentration available at the time, as applicable), replenishment of the food jars was documented.
- food consumption was not tabulated after lactation day 14, when it was expected that pups would begin to consume maternal food.

FURTHER OBSERVATIONS:
- duration of gestation: duration of gestation was calculated from gestation day 0 to the day the first pup was observed.
- parturition: apparently pregnant females were observed frequently from the expected day of parturition to determine, where possible, onset and duration of parturition.
- number of implantation sites

3) F1 GENERATION (producing F2 generation) (P1)

CAGE SIDE OBSERVATIONS: Yes
Time schedule:
- general appearance:
males: at least weekly and on the day of scheduled euthanasia
females: at least weekly, on gestation days 0, 7, 10, 14, and 20, and lactation days 0, 4, 7, 14, and 21, and the day of scheduled euthanasia

BODY WEIGHT: Yes
Time schedule for examinations:
- males: individually weighed at least weekly and on the day of scheduled euthanasia.
- females: individually weighed at least weekly, on gestation days 0, 7, 10, 14, 20, and 25 (as applicable) and lactation days 0, 4, 7, 14, and 21, at least weekly thereafter, and on the day of scheduled euthanasia

FOOD CONSUMPTION: Yes
- males: at least weekly during the exposure period
- females: at least weekly during the exposure period, on gestation days 0, 7, 10, 14, 18 and 20, and lactation days 0, 4, 7, 10 and 14.
- during cohabitation, when two rats occupied the same nesting box with one food jar, replenishment of the food jars was documented.
- food consumption was not tabulated after lactation day 14, when it was expected that pups would begin to consume maternal food.

SEXUAL MATURATION
- sexual maturation was evaluated daily until the criterion was achieved.
- females: observation for vaginal opening began on Day 27 postpartum.
- males: observation for preputial separation began on Day 34 postpartum.
- body weights were recorded on the day the criterion was achieved.

Oestrous cyclicity (parental animals):

Parental (P) generation (P0) and F1 generation (producing the F2 generation) (P1):
Oestrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Efforts to avoid inducing false pregnancy signs were performed during the daily procedure. Samples were collected for 14 consecutive days before pairing, during the cohabitation period until spermatozoa are observed in a smear of the vaginal contents and/or a copulatory plug is observed in situ, and on the day of scheduled euthanasia.

Sperm parameters (parental animals):

Parameters examined in P (P0) and F1 (P1) male generations:
- sperm motility (sperm from each vas deferens)
- sperm concentration in left cauda epididymis (sperm per gram of tissue weight)
- sperm morphology in portion of left cauda epididymides (determination of the percentage of normal sperm in a sample of at least 200, where feasible; and qualitative evaluation of abnormal sperm)
- spermatid counts (left testis; spermatids/gram of tissue weight)

Litter observations:

STANDARDISATION OF LITTERS (F1 and F2 generations)
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring:
- pup weight (F1 generation: days 0 (birth), 4, 7, 14 and 21 postpartum; F2 generation: days 0 (birth), 4, 7, 14, 21, 22, 23, and 24 postpartum)
- litter size
- viability at birth (number of live and dead pups at birth)
- postnatal mortality (at least twice daily during the preweaning period)
- number of pups in each litter (once daily during the preweaning period)
- clinical observations (daily during the preweaning period)
- developmental landmarks (pinna unfolding (day initated: day 2 postpartum), hair growth (day initiated: day 7 postpartum), incisor eruption (day initated: day 9 postpartum), eye opening (day initiated: day 12 postpartum), anogenital distance (once on day 4 postpartum (F2 only)); testing continued until the day the criterion was attained by all pups in the litter or until the day of scheduled euthanasia.)
- clinical chemistry: blood samples were collected from 2 pups/sex/litter (when possible) on Day 23 ± 2 days postpartum from the F1 and F2 generation pups not selected for continued observation, as applicable. Serum samples were analyzed for concentration of molybdenum and other elements.

GROSS EXAMINATION OF DEAD PUPS (F1 and F2 generation):
- Day 0 to 21 postpartum: pups that were found dead before examination of the litter for pup viability (Day 0 postpartum) were evaluated for vital status at birth. The lungs were removed and immersed in water. Pups with lungs that sank were identified as stillborn; pups with lungs that floated were identified as liveborn and to have died shortly after birth.
Pups that died (Days 1 to 21 postpartum) or were euthanized (Days 0 to 21 postpartum) before scheduled termination were examined for gross lesions and the cause of death or condition as soon as possible after the observation was made. The presence or absence of milk in the stomach was determined.

Postmortem examinations (parental animals):

1) PARENTAL (P) GENERATION (P0)
SACRIFICE
- Male animals: study days 147 to 151
- Maternal animals: lactation days 58 to 64
Animals that died or were euthanized before scheduled termination were examined for the cause of death or condition as soon as possible after the observation was made. The rats were examined for gross lesions, and a gross necropsy of the thoracic, abdominal, and pelvic viscera was performed.
Pregnancy status and uterine contents of female rats were recorded. Conceptuses in utero were examined to the extent possible as appropriate.

OVARIAN AND UTERINE EXAMINATIONS
- reproductive tract was dissected from the abdominal cavity.
- number and distribution of implantation sites were recorded.
- uteri of apparently nonpregnant rats were examined to confirm the absence of implantation sites

GROSS NECROPSY
- a gross necropsy of the thoracic, abdominal and pelvic viscera was performed at scheduled euthanasia and for all rats that were found dead or euthanized prior to scheduled termination.

ORGAN WEIGHTS
- the following organs were weighed at necropsy for all scheduled euthanasia animals: brain, epididymis, left cauda of epididymis, adrenal gland, coagulating gland, pituitary gland, prostate, seminal vesicles, thyroid, kidney, liver, ovaries, oviduct, spleen, thymus, testes, uterus
- organ weights were not recorded for animals found dead or euthanized in poor condition or in extremis.
- paired organs were weighed together, unless otherwise noted.
- organ weight as a percent of body weight (using the terminal body weight) was calculated.

HISTOPATHOLOGY
- the following representative samples of the tissues were collected from all animals and preserved: brain, cervix, epididymis, left cauda of epididymis, adrenal gland, coagulating gland, parathyroid, pituitary gland, prostate, seminal vesicles, thyroid, gross lesions (masses), kidney, liver, lungs, ovaries, oviduct, spleen, thymus, testes, uterus
- the following tissues were collected, processed, embedded, sectioned, and stained for microscopic evaluation: cervix, epididymis, left cauda of epididymis, adrenal gland, coagulating gland, parathyroid, pituitary gland, prostate, seminal vesicles, thyroid, gross lesions (masses), kidney, lungs, ovaries, oviduct, testes, and uterus
- entire left and right ovaries were embedded in separate paraffin blocks. Each ovary was sectioned beginning at 200 microns within the ovary. The first section was collected and designated as section 1 for follicle counts. After advancing the microtome 100 microns, the next section was collected as section 2. This process continued until a total of five sections from each ovary were collected for follicle counts, plus one section collected (between follicle count sections 3 and 4) on a separate slide for routine histopathological evaluation. The five sections were placed on one slide for each ovary. Section 1 was placed closest to the slide label, continuing in a linear pattern,
until five sections were on the same slide (section 5 being most distal to the slide label).
- tissues processed for primordial follicles were retained for possible future analysis. Presence or absence of corpora lutea was recorded for each animal. Group means and standard deviations were calculated for each animal (left, right, and both ovaries combined). Where the sample size was appropriate (N> 2), group mean values were compared in order to detect any significant differences in the number of primordial follicles in left ovaries, right ovaries, and both ovaries combined. Treated groups evaluated were compared statistically against groups that received control article only. Data were subjected to the Kruskal-Wallis nonparametric statistical test. If a significant difference was determined at p< 0.05, data were further analyzed using the Wilcoxon (Mann-Whitney U) Test.

TISSUE ANALYSIS
- at euthanasia, the liver and right kidney were collected
- tissues were analyzed for concentration of molybdenum and other elements

2) F1 GENERATION (producing the F2 generation) (P1)

SACRIFICE
- Male animals: study days 159 to 164
- Maternal animals: lactation days 24 to 34
Postweaning (> Day 21 postpartum): the F1 generation animals which died or were euthanised before before scheduled termination were examined for the cause of death or condition as soon as possible after the observation was made. The rats were examined for gross lesions, and a gross necropsy of the thoracic, abdominal, and pelvic viscera was performed.
Pregnancy status and uterine contents of the female rat were recorded. Conceptuses in utero were examined to the extent possible, where applicable.

OVARIAN AND UTERINE EXAMINATIONS
- reproductive tract was dissected from the abdominal cavity.
- number and distribution of implantation sites were recorded.
- uteri of apparently nonpregnant rats were examined to confirm the absence of implantation sites

GROSS NECROPSY
- a gross necropsy of the thoracic, abdominal and pelvic viscera was performed at scheduled euthanasia and for all rats that were euthanized prior to scheduled termination.

ORGAN WEIGHTS
- brain, spleen, and thymus were weighed at necropsy from one arbitrarily selected pup not selected for further evaluation per sex/litter at scheduled euthanasia, when possible.
- the following organs were weighed at necropsy for all scheduled euthanasia animals: brain, epididymis, left cauda of epididymis, adrenal gland, pituitary, prostate, seminal vesicles, thyroid, kidney, liver, ovaries, spleen, thymus, testes, and uterus
- organ weights were not recorded for animals euthanized in poor condition or in extremis.
- paired organs were weighed together, unless otherwise noted.
- organ weight as a percent of body weight (using the terminal body weight) was calculated.

HISTOPATHOLOGY
- the following representative samples of the tissues were collected from all animals and preserved: brain, cervix, epididymis, left cauda of epididymis, adrenal gland, parathyroid, pituitary gland, prostate, seminal vesicles, thyroid, gross lesions (masses), kidney, liver, lungs, ovaries, oviduct, spleen, thymus, testes, and uterus
- one control group animal/sex was selected from which all tissues examined at necropsy were retained, in order to provide control tissues for any possible histopathological evaluations of gross lesions.
- the following tissues were collected, processed, embedded, sectioned, and stained for microscopic evaluation: brain, cervix, epididymis, left cauda of epididymis, adrenal gland, parathyroid, pituitary gland, prostate, seminal vesicles, thyroid, gross lesions (masses), kidney, lungs, ovaries, oviduct, spleen, testes, and uterus
- entire left and right ovaries were embedded in separate paraffin blocks. Each ovary was sectioned beginning at 200 microns within the ovary. The first section was collected and designated as section 1 for follicle counts. After advancing the microtome 100 microns, the next section was collected as section 2. This process continued until a total of five sections from each ovary were collected for follicle counts, plus one section collected (between follicle count sections 3 and 4) on a separate slide for routine histopathological evaluation. The five sections were placed on one slide for each ovary. Section 1 was placed closest to the slide label, continuing in a linear pattern, until five sections were on the same slide (section 5 being most distal to the slide label).
- tissues processed for primordial follicles were retained for possible future analysis. Presence or absence of corpora lutea was recorded for each animal. Group means and standard deviations were calculated for each animal (left, right, and both ovaries combined). Where the sample size was appropriate (N> 2), group mean values were compared in order to detect any significant differences in the number of primordial follicles in left ovaries, right ovaries, and both ovaries combined. Treated groups evaluated were compared statistically against groups that received control article only. Data were subjected to the Kruskal-Wallis nonparametric statistical test. If a significant difference was determined at p< 0.05, data were further analyzed using the Wilcoxon (Mann-Whitney U) Test.

Postmortem examinations (offspring):

1) F1 GENERATION (pups not selected for continued observation)
SACRIFICE / GROSS NECROPSY
- all pups not selected for continued observation on Day 24 ± 2 days postpartum were euthanized and examined for gross lesions
- gross lesions were preserved.
- for one randomly-selected pup/sex/litter, the brain, spleen, thymus, thyroid, ovaries and uterus or testes and epididymides were preserved for possible future analysis.
- carcasses were discarded without further evaluation.

HISTOPATHOLOGY (pups not selected for continued observation)
The following tissues were identified for microscopic evaluation (target tissues) from 1 pup/sex/litter (when possible): brain, cervix, epididymis, left cauda of epididymis, adrenal gland, parathyroid, pituitary gland, prostate, seminal vesicles, thyroid, gross lesions (masses), kidney, lungs, ovaries, oviduct, spleen, testes, and uterus

2) F2 GENERATION
SACRIFICE / GROSS NECROPSY / ORGAN WEIGHTS
- on Day 24 ± 2 days postpartum, a gross necropsy of the thoracic, abdominal and pelvic viscera was performed for all pups.
- pups with gross lesions were preserved
- in addition, the following tissues were weighed/retained for 1 pup/sex/litter (when possible): brain, epididymis, adrenal gland, pituitary gland, prostate, seminal vesicles, thyroid, kidney, liver, ovaries, spleen, thymus, testes, and uterus
- organ weights were not recorded for animals euthanized in poor condition or in extremis.
- paired organs were weighed together, unless otherwise noted.
- all other tissues/carcasses were discarded

HISTOPATHOLOGY
- samples of the following tissues were collected from 1 arbitrarily selected pup/sex/litter (when possible) and all pups with gross lesions and preserved: brain, cervix, epididymis, adrenal gland, pituitary gland, prostate, seminal vesicles, thyroid, gross lesions/ masses, kidney, liver, lungs, ovaries, oviduct, spleen, thymus, testes, and uterus
- tissues identified for microscopic evaluation (target tissues) were examined from all pups with gross lesions: cervix, epididymis, adrenal gland, pituitary gland, prostate, seminal vesicles, thyroid, gross lesions/ masses, kidney, lungs, ovaries, oviduct, testes, and uterus

3) F1 GENERATION / F2 GENERATION
TISSUE ANALYSIS
- at euthanasia, the liver and right kidney were collected
- tissues were analyzed for concentration of molybdenum and other elements

Statistics:

Means, SD & % were calculated, as appropriate. Adult data were evaluated with the individual rat as the unit measured. Litter values were used in evaluation of pup data, as appropriate.
Clinical observation incidence data, & other proportional data were analyzed as contingency tables using the Fisher's Exact Test (1)*.
Continuous data, incl. variables with interval or ratio scales of measurement, such as body weights, food consumption & % motility were analyzed as follows:
Bartlett's Test of Homogeneity of Variances (2)* was used to test the hypothesis that all dose groups had equal variance. A nonsig. result (p>0.001) indicated that an assumption of homogeneity of variance was appropriate, & the data were compared using the ANOVA (3)*. If that test was sig. (p≤ 0.05), the groups given the test substance were compared with the control group using Dunnett's Test (4)*. If Bartlett's Test was sig. (p≤ 0.001), the ANOVA was not appropriate, & the data were analyzed as follows:
The Kruskal-Wallis Test was used to analyze the data, & in the event of a sig. result (p≤ 0.05), Dunn's Test (5)* was used to compare the groups given the test item with the control group. Count data, such as days in cohabitation, day a developmental landmark appears or No. of implantation sites, were analyzed using the Kruskal-Wallis Test & in the event of a sig. result (p≤ 0.05), Dunn's Test was used to compare the groups given the test item with the control group.
*References:
1) Siegel S. Nonparametric statistics for the behavioral sciences. New York (NY): McGraw-Hill Co; 1956. p. 96-105.
2) Sokal RR, Rohlf FJ. Biometry: the principles and practice of statistics in biological research. San Francisco (CA): Freeman & Co; 1969. p. 370-1.
3) Snedecor GW, Cochran WG. Statistical methods. 6th Ed. Iowa State University Press, Ames; 1967. p. 258-98.
4) Dunnett CW. J Am Stat Assoc 1955;50:1096-121.
5) Dunn OJ. Technometrics 1964;6(3):241-52.

Reproductive indices:

- mating index: percentage of pairings that resulted in matings
- fertility Index: percentage of matings that resulted in pregnancies
- gestation Index: Percentage of pregnancies that resulted in birth of live litters

Offspring viability indices:

- live birth index: percentage of pups born alive
- viability Indices: percentage of pups alive day 0 postpartum that survived to 3 days postpartum
- lactation Index: percentage of pups alive day 4 that survived 21 days postpartum

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- 40 mg Mo/kg bw/day: significantly increased (p≤ 0.05) number of male rats (7/24) compared to the untreated control group (1/24) had vocalization to touch. However any toxicological significance of this is unclear

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Effects that are considered as adverse and/or treatment related are described in the individual fields above.
Effects that are considered as non-adverse and/or not treatment-related are described in this field "Details on results".

CLINICAL SIGNS
40 mg Mo/kg bw/day: significantly increased (p≤ 0.05) number of male rats (7/24) compared to the untreated control group (1/24) had vocalization to touch. However, any toxicological significance of this is unclear. All other observed clinical observations in males and females were considered unrelated to the test substance (incidences were not dosage-dependent; observations occurred in only one rat; and/or observation is common in this species and strain), which included: chromorhinorrhea, sparse hair coat, a scab, swollen or torn pinna, localized alopecia, hunched posture, ungroomed coat, mass in the axilla, urine-stained abdominal fur, limited use of a limb, thin body condition and mild dehydration.

MORTALITY
5, 17, and 40 mg Mo/kg bw/day: no deaths considered related to sodium molybdate dihydrate occurred in the P generation.
Control group: one female rat was euthanized due to adverse clinical observations on day 23 of lactation. These clinical observations all occurred on lactation day 23 and included limited use of the right forelimb (limb with swelling in the axillary region), mild dehydration, hunched posture and thin body condition. The rat was apparently injured on lactation day 23. Body weight, food and water consumption during lactation demonstrated normal fluctuations. This dam had a normal litter of 18 pups all of which survived to weaning. There was a small thymus at necropsy that correlated, microscopically, with mild decreased cellularity. Additional microscopic findings included minimal infiltrates of histiocytes in the lung and mild pigmented macrophages in the uterus were considered incidental. The swelling in the right axillary region correlated, microscopically, with neutrophilic inflammation in the mammary gland tissue and the small thymus seen at necropsy correlated with mild decreased cellularity.
- 40 mg Mo/kg bw/day: one male rat was found dead on study day 62. This rat had no adverse clinical observations prior to death and was gaining weight prior to death. Water consumption was reduced on the day prior to death. Food consumption was comparable to other rats in the group. All tissues appeared normal at necropsy. Microscopically, there was moderate inflammation (mixed) in the lungs with foreign material (probably bedding material) and a mild infiltrate of mixed inflammatory cells in the prostate. The moderate inflammation seen in the lungs was considered likely related to aspiration and the cause of death of this animal.

BODY WEIGHT AND WEIGHT CHANGES
1) males
- 5, 17, and 40 mg Mo/kg bw/day: exposure to the test item had no effect on body weights or body weight gain.
- 40 mg Mo/kg bw/day: body weights were generally comparable to the concurrent control group for each weekly interval from study day 8 to 71 (last weight prior to cohabitation).
- 5, 17 and 40 mg Mo/kg bw/day: on study day 71, body weights were 98.8%, 98.4% and 98.4 % of the control group value, respectively. Body weights continued to be comparable to the control group value until euthanasia. On study day 143 weights were 98.8%, 99.2% and 97.2% of the control group value for the 5, 17 and 40 mg Mo/kg bw/day dose groups, respectively.
- no significant differences in body weight gain occurred with the exception of study day 57 to 64 when a significant increase (p≤ 0.05) in body weight gain occurred in the 5 and 40 mg Mo/kg bw/day dose groups (differences were not considered related to the test substance because the change did not persist or was not dose dependent).
2) females
- 5, 17, and 40 mg Mo/kg bw/day: body weights were generally comparable among the groups for each week during the premating, gestation and lactation periods.
- 5, 17, and 40 mg Mo/kg bw/day: on study day 71, body weights were 100.7%, 104.0% and 101.6% of the control group value, respectively.
- no significant differences in body weight or body weight gain occurred with the exception of significantly increased (p≤ 0.05 to p≤ 0.01) body weights in the 17 mg Mo/kg bw/day dose group for study days 36, 43 and 50; significantly increased (p≤ 0.05) body weight gains during the premating period in the 17 mg Mo/kg bw/day dose group for study days 22 to 29 and 36 to 43; and during the gestation period in the 17 mg Mo/kg bw/day dose group on days 10 to 14 of gestation. These differences were not considered related to the test substance because they were transient.

FOOD CONSUMPTION:
1) males
- 5, 17, and 40 mg Mo/kg bw/day: exposure to the test item had no effect on food consumption.
- 5, 17, and 40 mg Mo/kg bw/day: average food consumption was generally comparable among the groups for study days 1 to 71 and 1 to 143 compared to the control group values. Within this study interval, significantly reduced (p≤ 0.05 to p≤ 0.01) food consumption occurred in the 5 mg Mo/kg bw/day dose group on study days 22 to 29, 29, to 36 and 36 to 43; in the 17 mg Mo/kg bw/day dose group on study days 22 to 29 and 29 to 36 and in the 40 mg Mo/kg bw/day dose group on study days 120 to 122. These statistically significant differences were not considered related to the test substance because these differences were transient.
2) females
- 5, 17, and 40 mg Mo/kg bw/day: average food consumption was comparable among the groups with no statistically significant differences occurring for study days 1 to 71, gestation days 0 to 20 and lactation days 0 to 14 and for any interval evaluated compared to the control group value for the premating, gestation and lactation periods.

WATER CONSUMPTION
1) males
- 5, 17, and 40 mg Mo/kg bw/day: exposure to the test item at concentrations had no effect on water consumption.
- 40 mg Mo/kg bw/day: average water consumption was generally comparable among the groups for study days 1 to 71 and 1 to 143 in the 40 mg Mo/kg bw/day dose group compared to the control group value.
- 5, 17 and 40 mg Mo/kg bw/day: average water consumption for study days 1 to 71 was 101.8%, 105.9% and 99.8% in the 5, 17 and 40 mg Mo/kg bw/day dose groups compared to the control group value, respectively and 101.1%, 105.4% and 98.2% of the control group value for study days 1 to 143. Average water consumption was significantly increased (p≤ 0.05 to p≤ 0.01) for DS 1 to 8 in the 5, 17 and 40 mg Mo/kg bw/day dose groups compared to the control group value.
2) females
- 5, 17 and 40 mg Mo/kg bw/day: average water consumption was generally comparable among the groups for study days 1 to 71, gestation days 0 to 20 and lactation days 0 to 14 compared to the control group values.
- 5, 17 and 40 mg Mo/kg bw/day: average water consumption for study days 1 to 71 was 102.0%, 97.2% and 97.7% of the control group value for the 5, 17 and 40 mg Mo/kg bw/day dose groups, respectively, for gestation days 0 to 20, 97.1%, 95.6% and 98.0% and for lactation days 0 to 14, 99.1%, 100.9%, and 100.4% for the same respective dose groups.

COMPOUND INTAKE (Via Water)
Dose Level in mg Mo/kg bw/day in Drinking Water

1) Males
Dose Level (mg Mo/kg bw/day) 5 17 40
Water Water Water
Males
Lowest Average Value 4.6 15.0 34.2
Highest Average Value 5.9 18.7 44.8
Average 5.1 ± 0.4 18.0 ± 1.3 39.7 ± 3.2


5 mg Mo/kg bw/day:
- lowest average daily consumed molybdenum dose: 4.4 mg Mo/kg bw/day
- highest average daily consumed molybdenum dose: : 5.9 mg Mo/kg bw/day
- Average daily consumed molybdenum dose: 5.1 mg Mo/kg bw/day
17 mg Mo/kg bw/day:
- lowest average daily consumed molybdenum dose: 16.3 mg Mo/kg bw/day
- highest average daily consumed molybdenum dose: 21.0 mg Mo/kg bw/day
- Average daily consumed molybdenum dose: 18.0 mg Mo/kg bw/day
40 mg Mo/kg bw/day:
- lowest average daily consumed molybdenum dose: 35.2 mg Mo/kg bw/day
- highest average daily consumed molybdenum dose: 46.1 mg Mo/kg bw/day
- Average daily consumed molybdenum dose: 39.7 mg Mo/kg bw/day

2) Females
Dose Level (mg Mo/kg bw/day) 5 17 40
Water Water Water
Precohabitation
Lowest Average Value 4.6 15.0 34.2
Highest Average Value 5.9 18.7 44.8
Average 5.4±0.4 17.0 ± 1.2 41.1 ± 3.5
Gestation
Lowest Average Value 4.4 15.0 35.6
Highest Average Value 6.0 19.6 47.6
Average 5.1± 0.7 16.9±2.0 42.0 ± 5.1
Lactation
Lowest Average Value 5.0 17.0 41.0
Highest Average Value 6.6 22.7 67.4
Average 5.9±0.8 20.0 ± 2.9 49.4± 9.5


2) females
Precohabitation
5 mg Mo/kg bw/day (precohabitation):
- lowest average daily consumed molybdenum dose: 4.6 mg Mo/kg bw/day
- highest average daily consumed molybdenum dose: 5.4 mg Mo/kg bw/day
- Average daily consumed molybdenum dose: 5.1 mg Mo/kg bw/day
17 mg Mo/kg bw/day (precohabitation):
- lowest average daily consumed molybdenum dose 15.0 mg Mo/kg bw/day
- highest average daily consumed molybdenum dose: 18.7 mg Mo/kg bw/day
- Average daily consumed molybdenum dose: 17.0 mg Mo/kg bw/day
40 mg Mo/kg bw/day (precohabitation):
- lowest average daily consumed molybdenum dose: 34.2 mg Mo/kg bw/day
- highest average daily consumed molybdenum dose: 44.8 mg Mo/kg bw/day
- Average daily consumed molybdenum dose: 41.1 mg Mo/kg bw/day

Gestation
5 mg Mo/kg bw/day (gestation):
- lowest average daily consumed molybdenum dose: 4.4 mg Mo/kg bw/day
- highest average daily consumed molybdenum dose: 6.0 mg Mo/kg bw/day
- Average daily consumed molybdenum dose: 5.1 mg Mo/kg bw/day
17 mg Mo/kg bw/day (gestation):
- lowest average daily consumed molybdenum dose: 15.0 mg Mo/kg bw/day
- highest average daily consumed molybdenum dose: 19.6 mg Mo/kg bw/day
- Average daily consumed molybdenum dose: 16.9 mg Mo/kg bw/day
40 mg Mo/kg bw/day (gestation):
- lowest average daily consumed molybdenum dose: 35.6 mg Mo/kg bw/day
- highest average daily consumed molybdenum dose: 47.6 mg Mo/kg bw/day
- Average daily consumed molybdenum dose: 49.4 mg Mo/kg bw/day

Lactation
5 mg Mo/kg bw/day (lactation):
- lowest average daily consumed molybdenum dose: 5.0 mg Mo/kg bw/day
- highest average daily consumed molybdenum dose: 6.6 mg Mo/kg bw/day
- Average daily consumed molybdenum dose: 5.9 mg Mo/kg bw/day
17 mg Mo/kg bw/day (lactation):
- lowest average daily consumed molybdenum dose: 17.0 mg Mo/kg bw/day
- highest average daily consumed molybdenum dose: 22.7 mg Mo/kg bw/day
- Average daily consumed molybdenum dose: 20.0 mg Mo/kg bw/day
40 mg Mo/kg bw/day (lactation):
- lowest average daily consumed molybdenum dose: 41.0 mg Mo/kg bw/day
- highest average daily consumed molybdenum dose: 67.4 mg Mo/kg bw/day
- Average daily consumed molybdenum dose: 54.0 mg Mo/kg bw/day

ORGAN WEIGHT FINDINGS INCLUDING ORGAN / BODY WEIGHT RATIOS
1) males
- 0, 5, 17 and 40 mg Mo/kg bw/day: terminal body weights did not differ significantly among the groups. Organ weights and the ratio of these weights were not affected by the test item at doses as high as 40 mg Mo/kg bw/day in the water.
`- 40 mg Mo/kg bw/day: absolute weight of the liver and the ratio of the liver weight to the terminal body weight were significantly reduced (p≤ 0.05) compared to the control group values. These reductions were not considered adverse because the reductions were less than 10% of the control group value and there was no adverse histopathology in this group. When group sizes are 20 and greater numerical differences of less than 10% are often statistically significant and probably represent normal variation between groups especially when no other indicators of toxicity are occurring.
2) females
- 0, 5, 17 and 40 mg Mo/kg bw/day: terminal body weight was comparable among the groups.
- 17 and 40 Mo/kg bw/day: absolute weight of the thyroid/parathyroid and ratio of this organ weight to the terminal body weight were significantly reduced (p≤ 0.05 to p≤ 0.01) proportionate to dosage, but were considered not adverse as there were no histopathological changes and no difference was apparent in the females at 40 mg/kg/day in the diet. . The difference in organ weight or the ratio of the organ weight to the terminal body weight was not considered adverse because no histopathological changes occurred in the organ. The differences in the thyroid/parathyroid were greater than 10% but this probably reflected a greater variability in trimming and weighing this small tissue.


GROSS PATHOLOGICAL FINDINGS
1) Males and Females
- 5, 17, and 40 mg Mo/kg bw/day: no gross lesions related to sodium molybdate dihydrate at doses as high as 40 mg Mo/kg bw/day water occurred.
- 0 mg Mo/kg bw/day: one male had a small epididymides and small and flaccid testes. One non-pregnant female had a uterus that was diagnosed with pyometra with 15 to 20 mL of cloudy fluid (normal finding for a cycling non-pregnant rat). Another female had a small thymus.
- 5 mg Mo/kg bw/day: two males had a firm tan lobular mass in the epididymis (cut surface revealed a clear fluid) and one of these males did not sire a litter. A third male had two tan raised areas on the prostate
- 17 mg Mo/kg bw/day: one male had numerous tan raised areas on the prostate. Another male had small testes and epididymides. This was considered an incidental finding and although this animal mated, it did not sire a litter. One female had a tan mass in the left inguinal area, upon which the cut surface revealed a tan firm material.
- 40 mg Mo/kg bw/day: one male had a larger heart

HISTOPATHOLOGICAL FINDINGS - NON-NEOPLASTIC
The histological findings observed in the organs were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to ingestion of the test material.
1) Males
- 5, 17, and 40 mg Mo/kg bw/day: no test item-related microscopic findings were noted. Minimal or mild degeneration of the seminiferous tubules was observed in 1 of 24 rats in the 17 mg Mo/kg bw/day water dosed group. Severe degeneration of the seminiferous tubules was observed in an additional rat in the 17 mg Mo/kg bw/day water dosed group.

2) Females
- 17 mg Mo/kg bw/day: a statistically significant increase was noted in the number of primordial follicles in left ovaries compared to the control group. Since there was no significance in the right ovaries or total count for both left and right ovaries, and the slight increase observed in the treatment group versus the control group was not indicative of a clear pattern or trend, the changes observed were most likely due to natural variability among animals, and not to administration of the test item in the adult female rats.
The increased primordial follicles counts were not considered toxicologically important or adverse because: 1) there was no effect on fertility in either generation; 2) there was no treatment related observations on the histologic evaluation of the ovaries from the high dose group animals i.e., orderly maturation of the primary follicles to the subsequent stages, 3) the values were all within the historical control range and 4) no effect on ovarian weight or the ratio of these weights to the terminal body weight.

REPRODUCTIVE FUNCTION: OESTRUS CYCLE
- 5, 17, and 40 mg Mo/kg bw/day: exposure did not affect premating oestrous cycling.

REPRODUCTIVE FUNCTION: SPERM MEASURES
- 5, 17, and 40 mg Mo/kg bw/day: exposure to sodium molybdate dihydrate of up to 40 mg Mo/kg bw/day in water did not affect sperm motility, concentration or morphology

REPRODUCTIVE PERFORMANCE
- 5, 17, and 40 mg Mo/kg bw/day: exposure did not affect mating or fertility of the male or female rats. All mating and fertility parameters (rats that mated, fertility index, rats with confirmed mating dates during the first week of cohabitation and rats inseminated per rats in cohabitation) were comparable among the groups.

- 5, 17, and 40 mg Mo/kg bw/day: all rats mated (exceptions: 0 mg/kg bw/day: one female; 5 mg Mo/kg bw/day: one female). A total of 22 (95.6%), 22 (95.6%), 23 (95.8%) and 22 (95.6%) of the 24 male and female rats per group in the 0, 5, 17 and 40 mg Mo/kg bw/day exposure groups produced litters. The number of days in cohabitation was comparable among the groups.

- 5, 17, and 40 mg Mo/kg bw/day: natural delivery observations were unaffected by dosages of the test substance as high as 40 mg Mo/kg bw/day. Values for the numbers of dams delivering litters, the durations of gestation, averages for implantation sites per delivered litter, the gestation index, numbers of dams with stillborn and of dams with all pups dying were comparable among the four dosage groups and did not significantly differ.

BLOOD AND TISSUE ANALYSIS
- blood serum levels of molybdenum show a clear and dose related increase over the whole dose range, with the levels in males exceeding those in the females.
- females had a reduced 24 hour urinary excretion of molybdenum compared to males.
- liver and kidney levels of molybdenum was also increased in a dose related manner, with the highest levels being observed in the kidneys .

Key result

Dose descriptor:

LOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

equivalent to 40 mg Mo/kg bw/day

Sex:

male

Basis for effect level:

clinical signs

Remarks on result:

other:

Remarks:

Any toxicological significance in this effect is unclear

Key result

Dose descriptor:

NOAEL

Effect level:

42.5 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

equivalent to 17 mg Mo/kg bw/day

Sex:

male

Basis for effect level:

clinical signs

Remarks on result:

other:

Remarks:

Based on effects at 100 mg/kg bw/day

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Details on results:

NOTE: results of the F1 generation Postweaning are reported in this section since these animals were considered to be the second parental generation (P1)
Effects that are considered as adverse and/or treatment related are described in the individual fields above.
Effects that are considered as non-adverse and/or not treatment-related are described in this field "Details on results".
CLINICAL SIGNS
1) males
- 5, 17, and 40 mg Mo/kg bw/day: no clinical observations related to test item treatment occurred.
- all clinical observations were considered unrelated to the test substance (incidences were not dosage dependent; observations occurred in only one or two rats; observation occurred due to an injury; and/or observation is common in this species and strain), which included: ungroomed coat, vocalization to touch, spare hair coat, bent tail, mass in the axilla, and chromorhinorrhea.
2) females
- 5, 17, and 40 mg Mo/kg bw/day: no adverse clinical observations related to the test substance occurred during the premating, gestation or lactation periods.
- no observation occurred in a significantly increased number of rats in any group during any study period.
- all clinical observations were considered unrelated to the test substance (incidences were not dosage-dependent; observations occurred in only one rat; and/or observation is common in this species and strain), which included: vocalization to touch, spars hair coat, hunched posture, head tilt, urine-stained abdominal fur, and ungroomed coat.

MORTALITY
- 5, 17, and 40 mg Mo/kg bw/day: no deaths considered related to the test item occurred in the F1 generation.
The following early deaths occurred, but those were clearly not related to the test item:
- 0 mg Mo/kg bw/day: one male rat was euthanized on Day 158 postpartum due to adverse clinical observations. This rat had limited use of the hindlimbs during the 23rd week postpartum. The rat was gaining weight until Day 155 postpartum. Food and water consumption was comparable to other rats in the group. Necropsy revealed a flaccid testis. At necropsy, there was an abnormal appearance of the right testes that correlated with marked degeneration of the seminiferous tubules, microscopically. Also seen were mild hypospermatogenesis and minimal cellular debris in the epididymis and minimal nephropathy in the kidney. The cause of the acute hindlimb paralysis could not be determined by the gross and microscopic findings which were considered incidental.

- 17 mg Mo/kg bw/day: one male rat was euthanized due to adverse clinical observations on Day 44 postpartum. This rat had ungroomed coat four weeks postpartum and an enlarged eye in week seven postpartum. The rat was gaining weight through Day 43 postpartum. Food and water consumption was comparable to other rats in the group. The eye ruptured at necropsy. All other tissues appeared normal at necropsy. Microscopically in the left eye there was a marked corneal ulcer with neutrophilic inflammation in the corneal stroma, anterior chamber and the iris with fibrin and hemorrhage admixed and proliferation of the lens epithelium. Also seen was severe hypospermatogenesis in the epididymides which was considered incidental.
Furthermore, one female rat died during blood collection. At necropsy, there was an abnormal content (red gelatinous material) in the thoracic cavity and moderate hemorrhage in the lungs, microscopically, consistent with a blood collection-related accidental death.

BODY WEIGHT AND WEIGHT CHANGES
1) males
- 40 mg Mo/kg bw/day: body weights were generally comparable to the concurrent control group for each weekly interval from Day 22 to 106 postpartum (last weight prior to cohabitation). Body weight gains for Days 22 to 106 postpartum were significantly reduced (p≤ 0.05), 93.8% of the control group value. Although not significantly reduced, body weight gains for Days 22 to162 postpartum were 93.7% of the control group value.

- 5, 17 and 40 mg Mo/kg bw/day: on Day 106 postpartum, body weights were 99.8%, 95.8% and 95.2 % of the control group value for the 5, 17 and 40 mg Mo/kg bw/day dose groups, respectively. Body weights continued to be comparable to the control group value until euthanasia. On Day 162 postpartum weights were 100.4%, 96.6% and 94.9% of the control group value for the 5, 17 and 40 mg Mo/kg bw/day dose groups, respectively.

- no other significant differences in body weight gain occurred with the exception of Days 43 to 50 postpartum when a significant decrease (p≤ 0.05) in body weight gain occurred in the 40 mg Mo/kg bw/day dose group and on Days 64 to 71 postpartum when a significant decrease (p≤ 0.05) in body weight gain occurred in the 5 and 40 mg Mo/kg bw/day dose groups. These differences were not considered related to the test substance because the change did not persist or were not dose dependent.

2) females
- 5, 17 and 40 mg Mo/kg bw/day: body weights were generally comparable among the groups for each week during the premating, gestation and lactation periods. On Day 106 postpartum, body weights were 97.6%, 100.3% and 99.5% of the control group value for the 5, 17 and 40 mg Mo/kg bw/day dose groups, respectively.

- Statistically significant reductions and/or body weight losses (p≤ 0.05 to p≤ 0.01) occurred in the 40 mg Mo/kg bw/day dose group occurred for gestation body weight gain (gestation days 0 to 20), lactation days 21 and lactation days 14 to 21 and 0 to 21.

FOOD CONSUMPTION
1) males
- 5 mg Mo/kg bw/day: average food consumption was not affected by exposure.
- 17 and 40 mg Mo/kg bw/day: average food consumption was significantly reduced (p≤ 0.05 to p≤ 0.01) for each weekly interval from Days 57 to 64 to 99 to 106 postpartum compared to the control group value.
- 40 mg Mo/kg bw/day: statistically significant reductions in food consumption values on Days 127 to 134 and 141 to 148 postpartum occurred post cohabitation.
- 5, 17 and 40 mg Mo/kg bw/day: food consumption for Days 22 to 106 postpartum were 97.8%, 94.8% and 94.4% of the control group value for the 5, 17 and 40 mg Mo/kg bw/day dose groups, respectively. Food consumption for Days 22 to 162 postpartum were 98.2.%, 94.9% and 94.9% of the control group value for the 5, 17 and 40 mg Mo/kg bw/day dose groups, respectively.
2) females
- 5, 17 and 40 mg Mo/kg bw/day: average food consumption was comparable among the groups with no statistically significant differences occurring for Days 22 to 106 postpartum, gestation days 0 to 20 and lactation days 0 to 14 and for any interval evaluated compared to the control group value.

CONSUMED DOSES
All values were close to the target doses
1) males
5 mg Mo/kg bw/day:
- lowest average daily consumed molybdenum dose: 1.8 mg Mo kg bw/day
- highest average daily consumed molybdenum dose: 7.4 mg Mo kg bw/day
Average daily consumed molybdenum dose: 4.9 mg Mo kg bw/day

17 mg Mo/kg bw/day:
- lowest average daily consumed molybdenum dose: 6.2 mg Mo kg bw/day
- highest average daily consumed molybdenum dose: 27.3 mg Mo kg bw/day
Average daily consumed molybdenum dose: 17.4 mg Mo kg bw/day

40 mg Mo/kg bw/day:
- lowest average daily consumed molybdenum dose: 13.8 mg Mo kg bw/day
- highest average daily consumed molybdenum dose: 60.8 mg Mo kg bw/day
Average daily consumed molybdenum dose: 39.0 mg Mo kg bw/day

2) females
5 mg Mo/kg bw/day (precohabitation):
- lowest average daily consumed molybdenum dose: 1.7 mg Mo kg bw/day
- highest average daily consumed molybdenum dose: 7.4 mg Mo kg bw/day
Average daily consumed molybdenum dose: 45.0 mg Mo kg bw/day

17 mg Mo/kg bw/day (precohabitation):
- lowest average daily consumed molybdenum dose: 6.0 mg Mo kg bw/day
- highest average daily consumed molybdenum dose: 26.1 mg Mo kg bw/day
Average daily consumed molybdenum dose: 16.9 mg Mo kg bw/day

40 mg Mo/kg bw/day (precohabitation):
- lowest average daily consumed molybdenum dose: 14.0 mg Mo kg bw/day
- highest average daily consumed molybdenum dose: 60.1 mg Mo kg bw/day
Average daily consumed molybdenum dose: 40.3 mg Mo kg bw/day

5 mg Mo/kg bw/day (gestation):
- lowest average daily consumed molybdenum dose: 4.3 mg Mo kg bw/day
- highest average daily consumed molybdenum dose: 5.8 mg Mo kg bw/day
Average daily consumed molybdenum dose: 4.9 mg Mo kg bw/day

17 mg Mo/kg bw/day (gestation):
- lowest average daily consumed molybdenum dose: 13.8 mg Mo kg bw/day
- highest average daily consumed molybdenum dose: 19.4 mg Mo kg bw/day
Average daily consumed molybdenum dose: 16.3 mg Mo kg bw/day

40 mg Mo/kg bw/day (gestation):
- lowest average daily consumed molybdenum dose: 36.4 mg Mo kg bw/day
- highest average daily consumed molybdenum dose: 45.5 mg Mo kg bw/day
Average daily consumed molybdenum dose: 39.6 mg Mo kg bw/day

5 mg Mo/kg bw/day (lactation):
- lowest average daily consumed molybdenum dose: 5.2 mg Mo kg bw/day
- highest average daily consumed molybdenum dose: 6.6 mg Mo kg bw/day
Average daily consumed molybdenum dose: 6.0 mg Mo kg bw/day

17 mg Mo/kg bw/day (lactation):
- lowest average daily consumed molybdenum dose: 16.7 mg Mo kg bw/day
- highest average daily consumed molybdenum dose: 20.9 mg Mo kg bw/day
Average daily consumed molybdenum dose: 19.1 mg Mo kg bw/day

40 mg Mo/kg bw/day (lactation):
- lowest average daily consumed molybdenum dose: 41.3 mg Mo kg bw/day
- highest average daily consumed molybdenum dose: 50.0 mg Mo kg bw/day
Average daily consumed molybdenum dose: 146.7 mg Mo kg bw/day

WATER CONSUMPTION
1) males
- 5, 17 and 40 mg Mo/kg bw/day: average water consumption was generally comparable among the groups for Days 22 to 106 and 22 to 162 postpartum compared to the control group value. Average water consumption for Days 22 to 106 postpartum was 103%, 103% and 98.9% in the 5, 17 and 40 mg Mo/kg bw/day dose groups compared to the control group value, respectively and 101.5%, 99.8% and 96.2% of the control group value for Days 22 to 162 postpartum.
2) females
- 5, 17 and 40 mg Mo/kg bw/day: average water consumption was generally comparable among the groups for Days 22 to 106 postpartum and gestation days 0 to 20 compared to the control group values. Average water consumption for Days 22 to 106 postpartum was 99.1%, 101.1% and 103.7% of the control group value for the 5, 17 and 40 mg Mo/kg bw/day dose groups, respectively, for gestation days 0 to 20, 97.0%, 95.4% and 97.8% and for lactation days 0 to 14, 99.0%, 95.0% and 98.5% for the same respective dose groups.

ORGAN WEIGHT FINDINGS INCLUDING ORGAN / BODY WEIGHT RATIOS
1) males
- 0, 5, 17 and 40 mg Mo/kg bw/day: terminal body weights did not differ significantly (p≤ 0.05) among the 0, 5, 17 and 40 mg Mo/kg bw/day water dose groups. The terminal body weights were 99.8%, 96.2% and 94.6% of the control group value for the 5, 17 and 40 mg Mo/kg bw/day water dose groups, respectively.
- 17 mg Mo/kg bw/day: ratio of the weight of the left epididymis to the terminal body weight was significantly increased (p≤ 0.05) compared to the control group value (not considered adverse as the increase was not dose-dependent).
- 40 mg Mo/kg bw/day: absolute weight of the liver was significantly reduced (p≤ 0.05) compared to the control group values. The ratio of the left and right epididymis, the left testis minus the tunica albuginea, paired adrenals and thyroid/parathyroid to the terminal body weight were all significantly increased (p≤ 0.05 to p≤ 0.01) compared to the control group values. These significant changes in organ weights were not considered adverse as they reflected the slightly lower terminal body weights in these groups and no histopathology related to the test item occurred in these organs.
2) females
- 0, 5, 17 and 40 mg Mo/kg bw/day: terminal body weight was comparable among the groups. The absolute weights of the pituitary, brain, liver, kidneys, adrenals, spleen, thymus, ovaries, uterus and thyroid/parathyroid and the ratio of these organ weights to the terminal body weights were not affected by exposure in drinking water at up to 40 mg Mo/kg bw/day.

GROSS PATHOLOGICAL FINDINGS
- 5, 17 and 40 mg Mo/kg bw/day: no gross lesions related to sodium molybdate dihydrate at doses as high as 40 mg Mo/kg bw/day water occurred.

None of the gross lesions were considered related to the test item because the lesion was observed in control and treated animals, the incidence was not dose-dependent and there was no histopathological changes related to the test item in these organs in the water dosed group. None of these findings had any impact on overall fertility incidence. The following findings were recorded:
1) males
- 0 mg Mo/kg bw/day: one male rat had a flaccid testis. Another male rat had numerous red areas on the thymus and a firm yellow mass on the testis that when cut revealed a yellow caseous material. This male did not sire a litter.
- 5 mg Mo/kg bw/day: three male rats had slight dilation of the renal pelvis. Another male rat had clear fluid filled cysts in one kidney. A fifth male rat had red areas on the thymus. Lastly, one male rat had small epididymides and flaccid testes. This male mated but did not sire a litter.
- 17 mg Mo/kg bw/day: one male rat had slight dilation of the renal pelvis. Another male rat had numerous calculi in the urinary bladder that had thick walls. The ureters were dilated and renal pelvis had slight dilation with numerous pitted tan areas.
- 40 mg Mo/kg bw/day: two male rats had slight dilation of the renal pelvis. Both males mated but one, which also had unilateral small epididymis and testis, did not sire a litter. Another male rat had red areas on the thymus. A fourth male rat had a lobular mass (location not recorded) that was tan, red and firm.
2) females
- 0 mg Mo/kg bw/day: one female rat had red pinpoint areas on the thymus. This rat was never pregnant.
- 17 mg Mo/kg bw/day: one female rat had a red gelatinous material in the thorax. This rat died during blood collection.
- 40 mg Mo/kg bw/day: one female rat had slight dilation of the renal pelvis of each kidney and numerous clear fluid-filled cysts that extended into the parenchyma. This rat was never pregnant.

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC
- 5, 17 and 40 mg Mo/kg bw/day: no test item-related microscopic findings were noted.
- corpora lutea were present for all scheduled euthanasia animals evaluated in all groups in the F1 Generation.
- counts demonstrated normal variability in this strain of rat.
- values in the treated groups were all increased above the control group (not considered toxicologically important or adverse, because: no effect on fertility in either generation; no treatment related observations on the histologic evaluation of the ovaries from the high dose group animals i.e., orderly maturation of the primary follicles to the subsequent stages; values were all within the historical control range and no effect on ovarian weight or the ratio of these weights to the terminal body weight.)

BLOOD AND TISSUE ANALYSIS
- F1 adults also show a clear dose related increase in molybdenum levels in the serum over the whole dose range with the male levels exceeding the females
- liver and kidney levels of molybdenum were also increased in a dose related manner in the F1 adults, with the highest levels being observed in the kidneys.

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

REPRODUCTION FUNCTION: ESTRUS CYCLE
- 5, 17 and 40 mg Mo/kg bw/day: exposure to sodium molybdate dihydrate at doses of molybdenum as high as 40 mg Mo/kg bw/day in the water did not affect premating oestrous cycling.
REPRODUCTION FUNCTION: SPERM MEASURES
- 5, 17 and 40 mg Mo/kg bw/day: exposure to sodium molybdate dihydrate of up to 40 mg Mo/kg bw/day in water did not affect sperm motility, concentration or morphology. The percent of sperm with no head was not significantly different in any of the treated groups compared to the control value
REPRODUCTIVE PERFORMANCE
1) males and females
- 5, 17 and 40 mg Mo/kg bw/day: exposure to sodium molybdate dihydrate at doses as high as 40 mg Mo/kg bw/day in the water did not affect mating or fertility of the male or female rats. All mating and fertility parameters (rats that mated, fertility index, rats with confirmed mating dates during the first week of cohabitation and rats inseminated per rats in cohabitation) were comparable among the groups.
2) males
- 5, 17 and 40 mg Mo/kg bw/day: all male rats mated. Of these a total of 22/24 (91.7%), 23/24 (95.8%), 21/22 (95.4%) and 19/24 (79.2%) of the 24 /24 male rats per group in the 0, 5, 17 and 40 mg Mo/kg bw/day exposure groups produced litters. The number of days in cohabitation (2.3 to 3.0) was comparable among the groups.
3) females
- 5, 17 and 40 mg Mo/kg bw/day: a total of 22/24 (91.7%), 23/24(95.8%), 23/24 (95.8%) and 21/24 (87.5%) female rats per group in the 0, 5, 17 and 40 mg Mo/kg bw/day exposure groups produced litters. The number of days in cohabitation (2.4 to 3.3) was comparable among the groups.
Natural delivery observations were unaffected by dosages of the test substance as high as 40 mg Mo/kg bw/day. Values for the numbers of dams delivering litters, the durations of gestation, averages for implantation sites per delivered litter, the gestation index, numbers of dams with stillborn and of dams with all pups dying, were comparable among the four dosage groups and did not significantly differ.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 100 mg/kg bw/day

Based on:

element

Remarks:

equivalent to >= 40 mg Mo/kg bw/day

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Effects that are considered as adverse and/or treatment related are described in the individual fields above.
Effects that are considered as non-adverse and/or not treatment-related are described in this field "Details on results".

CLINICAL SIGNS:
- 5, 17, and 40 mg Mo/kg bw/day: no adverse clinical observations occurred in the pups at doses of the test substance as high as 40 mg Mo/kg bw/day.

No clinical observations considered related to the test item occurred as none occurred in more than one to three litters and/or the observation was not dose dependent. These observations included: cold to touch, a scab on the head, neck and/or base of tail, dehydration, ungroomed coat, not nesting, pale body, purple or black head or tip of tail, laceration, umbilical hernia, desheathed tail, tail, or portion of tail missing.

- 5, 17, and 40 mg Mo/kg bw/day: doses as high as 40 mg Mo/kg bw/day did not affect the onset or development of eye opening, hair growth, incisor eruption or pinna unfolding. No statistically significant differences occurred among the groups for any parameter evaluated.

MORTALITY / VIABILITY
- 0, 5, 17, and 40 mg Mo/kg bw/day: viability and lactation indices, and live litter size at weighing were comparable among the four dosage groups and did not significantly differ.

BODY WEIGHTS AND WEIGHT CHANGES
- 0, 5, 17, and 40 mg Mo/kg bw/day: values for the the pup weights were comparable among the four dosage groups and did not significantly differ.

GROSS PATHOLOGICAL FINDINGS
- 5, 17, and 40 mg Mo/kg bw/day: no adverse necropsy observations occurred in the pups at doses of the test substance as high as 40 mg Mo/kg bw/day.

No necropsy observations considered related to the test item occurred as none occurred in more than one to three litters and/or the observation was not dose dependent.
All pups necropsied on postnatal day 21 or 23 appeared normal except for two pups in a single 40 mg Mo/kg bw/day group litter that had dilation of the lateral ventricles of the brain. Necropsy of found dead pups revealed 1 to 2 pups in the 0, 5, 17 and 40 mg Mo/kg bw/day groups had no milk in their stomach. All other found dead pups appeared normal.

SEXUAL MATURATION
1) males
- 5, 17, and 40 mg Mo/kg bw/day: preputial separation and the average body weight of the male rats on the day of preputial separation were not affected by exposure to the test item at doses of molybdenum as high as 40 mg Mo/kg bw/day in the water.

2) females
- 5, 17, and 40 mg Mo/kg bw/day: vaginal patency and the average body weight of the female rats on the day of vaginal patency were not affected by exposure to the test item at doses of molybdenum as high as 40 mg Mo/kg bw/day in the water.

ORGAN WEIGHTS INCLUDING ORGAN / BODY WEIGHT RATIONS
- 5, 17, and 40 mg Mo/kg bw/day: no effect on organ weights (brain, spleen, and thymus) or ratio of the organ weight to the terminal body weight occurred in the pups at doses of the test substance as high as 40 mg Mo/kg bw/day.

HISTOPATHOLOGICAL FINDINGS
- 5, 17, and 40 mg Mo/kg bw/day: no test item-related microscopic findings were noted.

BLOOD AND TISSUE ANALYSIS
- dose related increase in serum levels of molybdenum are present in the F1 at weaning, but at lower levels than in the adults.

Key result

Dose descriptor:

NOAEL

Generation:

F1

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

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Effects that are considered as adverse and/or treatment related are described in the individual fields above.
Effects that are considered as non-adverse and/or not treatment-related are described in this field "Details on results".

CLINICAL SIGNS
- 5, 17, and 40 mg Mo/kg bw/day: no adverse clinical observations occurred in the pups at doses of the test substance as high as 40 mg Mo/kg bw/day.

- 5 mg Mo/kg bw/day: an increased number of litters (8) had pups with ungroomed coat and a significantly increased (p≤ 0.01) number of litters had pups with dehydration. These increases were not considered related to the test substance because the intergroup + incidences were not dose-dependent.

No other clinical observations considered related to sodium molybdate dihydrate occurred as none occurred in more than one to four litters and/or the observation was not dose dependent. These observations included: cold to touch, dehydration, laceration, purple or black area on the head, back, tip of tail or limb, pale, no milk band, a scab, microphthalmia, decreased motor activity, gasping, sparse hair coat, tail bent, ruptured eye.

- 5, 17, and 40 mg Mo/kg bw/day: maternal doses as high as 40 mg Mo/kg bw/day did not affect the onset or development of eye opening, hair growth, incisor eruption or pinna unfolding. No statistically significant differences occurred among the groups for any parameter evaluated.

MORTALITY / VIABILITY
- 0, 5, 17, and 40 mg Mo/kg bw/day: viability and lactation indices, and live litter size at weighing were comparable among the four dosage groups and did not significantly differ.

BODY WEIGHTS AND WEIGHT CHANGES
- 0, 5, 17, and 40 mg Mo/kg bw/day: values for the the pup weights were comparable among the four dosage groups and did not significantly differ.

SEXUAL MATURATION
- 5, 17, and 40 mg Mo/kg bw/day: anogenital distances for male and female pups on Day 4 postpartum did not differ among the groups in litters from dams with maternal doses as high as 40 mg Mo/kg bw/day.

ORGAN WEIGHT FINDINGS INCLUDING ORGAN / BODY WEIGHT RATIOS
- 5, 17, and 40 mg Mo/kg bw/day: no test substance-related effects on organ weights occurred in the pups at doses of the test substance as high as 40 mg Mo/kg bw/day.

- 5 mg Mo/kg bw/day: female absolute weights of the brain, spleen and thyroid/parathyroid were significantly reduced (p≤ 0.05 to p≤ 0.01) in comparison with the control group values. These reductions were not considered to be test substance related because they were not dose dependent.

GROSS PATHOLOGICAL FINDINGS
- 5, 17, and 40 mg Mo/kg bw/day: no treatment-related effects were observed in the pups at doses of the test substance as high as 40 mg Mo/kg bw/day.

- 0 , 5, 17, 40 Mo/kg bw/day: all pups necropsied on postnatal day 21 through 24 appeared normal except for 2, 1, 3, and 0 pups each from a different litter in the 0, 5, 17, 40 Mo/kg bw/day dosed water group pups, respectively. The pups in the control group had clear fluid filled cysts in the kidney, the pup in the 5 mg Mo/kg bw/day dosed water group had situs inversus of the abdominal viscera and two of three pups in the 17 mg Mo/kg bw/day dosed water group had slight dilation of renal pelvis and the third pup had a clear fluid filled cyst in the kidney.
Necropsy of found dead and stillborn pups revealed 2, 3, 2, and 0 in the 0, 5, 17 and 40 mg Mo/kg bw/day dosed water groups had no milk in their stomach. All other found dead or stillborn pups appeared normal.

HISTOPATHOLOGICAL FINDINGS - NON-NEOPLASTIC
- 5, 17, and 40 mg Mo/kg bw/day: no test item-related microscopic findings were noted.

- all the males and females were, as expected, sexually immature on postnatal day 22. In the males there was no active spermatogenesis in the testes and in the females no corpora lutea present in the ovaries in control or treated groups.

BLOOD AND TISSUE ANALYSIS
- dose related increase in serum levels of molybdenum are present in the F2 pups at weaning, but at lower levels than in the adults.

Key result

Dose descriptor:

NOAEL

Generation:

F2

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

Reproductive effects observed:

no

Conclusions:

Introductory remark:
The main driver for conducting this 2-generation study was for regulatory purposes in the United States of America. In the past, regulations deriving health thresholds for molybdenum have often relied on the non-guideline study by Fungwe et al. (1990) in which rats were exposed to molybdenum via drinking water, and US EPA were seeking a more robust study. Therefore, this 2-generation study was also conducted via drinking water, to see if it would be possible to replicate the findings reported by Fungwe et al. (1990). The range-finder and the main study each included three dose levels via drinking water. Since for humans the exposure via the diet is the more relevant route of exposure, the range-finder also included 3 dose levels via the diet. For the main study, only the top-dose was administered via the diet. The US EPA Office of Water was involved in the decision to select drinking water as the main route of administration in the main study.
For technical reasons, four separate study records had to be prepared in IUCLID for the drinking water and diet parts of the range-finder and main study, respectively. All parts of the study should be considered together.

This study record is for the main study, drinking water part of the study.

Summary:
The effects of sodium molybdate dihydrate on the male and female reproductive systems in Crl:CD (SD) Sprague Dawley rats, including gonadal function, oestrous cycles, mating behaviour, conception, gestation, parturition, lactation, weaning and the growth and development of offspring were investigated in an OECD Test Guideline 416 study. The dose levels were selected based on a previous sub-chronic (90 day) study, on a developmental toxicity study and on a rangefinder study in rats in which clear effects were observed on bodyweight at the top dose level of 40 mg Mo/kg bw/day. In the sub-chronic study, the top dose level of 60 mg Mo/kg bw/day produced excessive bodyweight loss in males, and renal toxicity in females. The dose levels for the 2 Generation Main study were 5,17 and 40 mg Mo/kg bw/day in drinking water and 40 mg Mo/kg bw/day via the diet.

The male rats were directly exposed to the test or control drinking water for at least 10 weeks before cohabitation, during the cohabitation, and continuing through to the day of euthanasia. The female rats were directly exposed to the test or control diet or drinking water for at least 10 weeks before cohabitation, during the cohabitation, gestation, littering and post-partum periods (lactation period; DL) and continuing through to the day of euthanasia.

F1 generation pups were directly exposed to the test and/or carrier control substances after they began consuming food/water (approximately on Day 15 postpartum). Pups will have been exposed to the test and/or carrier control substances during maternal gestation (in utero exposure) or via maternal milk during the lactation period. The selected F1 generation weaned pups were directly exposed to the test or control diet or drinking water at least 10 weeks before cohabitation (beginning on Day 22 postpartum), during the cohabitation, gestation, littering and post-partum periods (lactation period; DL) and continuing through to the day of euthanasia.
F2 generation pups were directly exposed to the test and/or carrier control substances after they begin consuming food/water (approximately on Day 15 postpartum). Pups will have been exposed to the test and/or carrier control substances during maternal gestation (in utero exposure) or via maternal milk during the lactation period.

Results:
All diet and water formulations that were analyzed were found to be within or very close to the planned quality control /quantity assurance parameters.
No deaths related to sodium molybdate dihydrate occurred in either the P or F1 generations. One P generation 40 mg Mo/kg bw/day drinking water group male rat was found dead on Study Day 62 (DS 62) which appeared to be secondary to aspiration of bedding material. In the P generation, one control group female rat was euthanised on Day 23 of lactation, and one 40 mg Mo/kg bw/day diet dose group female rat was euthanized on Day 43 of lactation due to adverse clinical observations (swollen limb, fractured palate) that appeared to be due to accidental injuries not related to sodium molybdate dihydrate.
One F1 generation control group male rat was euthanized on Day 158 postpartum and one F1 generation male rat in the 17 mg Mo/kg bw/day drinking water group was euthanized on Day 44 postpartum due to adverse clinical observations. One F1 generation female 17 mg Mo/kg bw/day drinking water dose group rat died during blood collection on Day 30 of lactation. All other weaned F1 generation rats survived to scheduled euthanasia.

Groups Exposed to the Test Material in the Drinking Water (0.5, 17 & 40 mg Mo/kg bw/day):
P Generation Male and Female Rats and F1 Generation to Weaning
Note in the IUCLID report results for the P Generation are recorded as P0 Generation
A significantly increased number of male rats in the 40 mg Mo/kg bw/day dose group had vocalization to touch compared with the controls. Any toxicological significance of this is unclear. No adverse clinical observations related to the test substance occurred during the premating, gestation or lactation periods at any dose levels.
No significant adverse effect was observed on P (P0) Generation male or female body weights or body weight gain. There was no effect on food or water consumption in males or females (for for the premating, gestation and lactation periods)
No effect on premating oestrous cycling, mating or fertility of the male or female rats was observed and total of 22, 22, 23 and 22 of the 24 female rats per group in the 0, 5, 17 and 40 mg Mo/kg bw/day exposure groups, respective and no adverse clinical or necropsy observations occurred in the F1 pups. In the FI pups, no effect was observed on the onset or development of eye opening, hair growth, incisor eruption or pinna unfolding. No effects on organ weights (brain, spleen, and thymus) or ratio of the organ weight to the terminal body weight occurred in the F1 pups.
Terminal body weights for the P (P0) Generation male and female rats did not differ significantly. The absolute weight of the liver in the males and the ratio of the liver weight to the terminal body weight were significantly reduced in the 40 mg Mo/kg bw/day male dose groups compared to the control group values. The absolute weight of the thyroid/parathyroid in females, and ratio of this organ weight to the terminal body weight were significantly reduced in the 17 and 40 Mo/kg bw/day female drinking water dosed groups compared to the control group values. These differences were not considered adverse because there were no histopathological changes in these organs and there was no effect on females receiving 40 mg/kg/day in the diet.
There was no effect on the P0-generation sperm motility, concentration or morphology.

F1 Generation Male and Female Rats raised for mating, and F2 Generation to Weaning
Note in the IUCLID report results for theF1 generation growth phase from weaning to mating are recorded as P1 generation'
F1 Generation Male and Female Rats raised for mating (F1 (P1) Generation)
No substance related adverse clinical observations occurred in the F1 (P1) Generation male or female rats. Body weights in F1 (P1) Generation male rats were generally comparable to the control group On Day 106 postpartum, body weights were 99.8%, 95.8% and 95.2 % of the control group value for the 5, 17 and 40 mg Mo/kg bw/day dose groups, respectively and on Day 162 postpartum weights were 100.4%, 96.6% and 94.9% of the control group. Body weight gains for Days 22 to 106 postpartum were significantly reduced (6.2%) in the 40 mg Mo/kg bw/day dose group compared to the control group values. Although not statistically significant, body weight gains for Days 22 to162 postpartum were also reduced (93.7%) compared with the control group value.
Body weights in F1 (P1) Generation female rats were generally comparable among the treated and control groups for each week during the premating period. On Day 106 postpartum (prior to mating), body weights were not significantly different at 97.6%, 100.3% and 99.5% of the control group value for the 5, 17 and 40 mg Mo/kg bw/day dose groups, respectively.
Significant reductions in body weight and/or body weight loss occurred in the 40 mg Mo/kg bw/day dose group during gestation (DGs 0 to 20, 9.7%,), and lactation, (DLs 0 21 and 14-21, mean weight loss of 4.5 g and 26.8 g compared with a gain in controls of 12.0 g).
Average food consumption in male rats was reduced or significantly reduced for each weekly interval from Days 56 to 106 postpartum in the 17 and 40 mg Mo/kg bw/day dose groups. Significant reduced food consumption occurred post cohabitation on Days 127 to 148 postpartum only in the 40 mg Mo/kg bw/day dose group.
Average food consumption in female rats was comparable among the groups with no significant differences occurring prior to mating, during gestation or lactation.
Average water consumption in male and female rats was generally comparable among the groups with no statistically significant differences occurring at any stage prior to mating, during gestation or lactation.
Preputial separation and vaginal patency and the average body weight on the day of sexual maturation were not affected and there was no effect on premating oestrous cycling, mating or fertility of the F1 male or female rats. A total of 22/24, 23/24, 21/22 and 19/24 female rats per group in the 0, 5, 17 and 40 mg Mo/kg bw/day produced litters.
The terminal body weights in male rats did not differ significantly among being were 99.8%, 96.2% and 94.6% of the control group value for the 5, 17 and 40 mg Mo/kg bw/day drinking water dosed groups, respectively. The absolute weight of the liver and the ratios of the left and right epididymis, the left testis minus the tunica albuginea, paired adrenals and thyroid/parathyroid to the terminal body weigh were significantly reduced in 40 mg Mo/kg bw/day male dose groups compared to the control group value. These effects were not considered adverse as they reflected the slightly lower terminal body weights in this exposure group.
Terminal body weight, organ weight and ratio of organ weight to the terminal body weight was not affected in adult female rats.
No treatment related microscopic findings were noted.
No effects were observed on sperm motility, concentration or morphology. Natural delivery was not affected.

F2-Pups
In the F2-pups no adverse clinical or necropsy observations were observed. No effect on the onset or development of eye opening, hair growth, incisor eruption or pinna unfolding and no effect on anogenital distances for male and female F2 pups on Day 4 postpartum.
No effect was observed on F2 pup organ weights (kidney, liver, thymus, pituitary, adrenals, testes, epididymides, prostate, seminal vesicles, ovaries, uterus) and no microscopic findings (F1/P1 adults, F2 pups) or effects on primordial follicle counts (F1/P1adults) occurred with the exception of absolute weight of the brain, spleen and thyroid/parathyroid for the females were significantly reduced only in 5 mg Mo/kg bw/day water dose group.

Serum and Tissue Levels in Water and Diet Dosed Groups:
Just prior to, and at scheduled euthanasia, urine, blood, liver and kidney samples were collected from P and F1 males and females for analysis for molybdenum and other element. Blood and tissue samples were also collected from F1 and F2 pups around Day 24 postpartum. The blood serum and tissue levels of molybdenum were consistently higher in the diet dosed animals compared with the drinking water dosed animals, and higher in the males than in the females in all groups.

Conclusion:
Based on the results of this study the no-observed-adverse-effect level (NOAEL) for paternal and maternal toxicity was 17 mg Mo/kg bw/day based on reductions in body weight and food consumption in the 40 mg Mo/kg bw/day dose groups (diet) see separate IUCLID RSS on 2-Generation Mian Study Diet Administration. The NOAEL for males and females of the P and F1 generations for reproductive toxicity, including mating and fertility and the growth and development of the F1 generations to adulthood and the growth and development up to weaning of the F2 generation, was 40 mg Mo/kg bw/day whether exposure occurred in the diet or drinking water.

The top dose level of 40 mg Mo/kg bw/day is approximately 20,000 times greater than the normal total dietary and water intake of molybdenum of 2 µg Mo/kg bw/day in humans (general population).

Executive summary:

In an OECD Test Guideline 416 multigenerational study, groups of 24 male and 24 female Sprague-Dawley rats were administered sodium molybdate dihydrate at 0, 5, 17, or 40 mg molybdenum (Mo)/kg bw/day in the drinking water and at 40 mg molybdenum (Mo)/kg bw/day in diet over two generations to assess reproductive toxicity. No adverse effect on reproductive function was observed at any dose level in either generation as indicated by no significant dose-related effect on oestrus cycles, sperm parameters, mating, fertility, gestation, litter size, pup survival, growth or postnatal development. Serum levels of molybdenum werer increased in a dose-related manner. The No Observed Adverse Effect Levels (NOAEL) are 17 mg Mo/kg bw/day for systemic toxicity and 40 mg Mo/kg bw/day for reproductive toxicity. The diet administration results are given in a separate IUCLID RSS entry.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

This dossier is one of several dossiers prepared under the auspices of the REACH Molybdenum Consortium (“MoCon”). To avoid unnecessary (animal) testing, a comprehensive grouping and read-across concept has been developed amongst several molybdenum containing substances. This grouping/category approach is described in detail in a separate report, in accordance with the ECHA's "Read-Across Assessment Framework" (RAAF). This document is attached to section 13 in the technical dosser and to the CSR. The following text in this paragraph is therefore applicable to all substances in the category (which are listed in the afore-mentioned report).

 

In an OECD Test Guideline 416 multigenerational study, groups of 24 male and 24 female Sprague-Dawley rats were administered sodium molybdate dihydrate at 0, 5, 17, or 40 mg molybdenum (Mo)/kg bw/day in the drinking water and at 40 mg molybdenum (Mo)/kg bw/day in diet over two generations to assess reproductive toxicity.

 

The main driver for conducting this 2-generation study was for regulatory purposes in the United States of America. In the past, US regulations deriving health thresholds for molybdenum have often relied on the non-guideline study by Fungwe et al. (1990) in which rats were exposed to molybdenum via drinking water, and US EPA were seeking a more robust study. Therefore, this 2-generation study was also conducted via drinking water, to see if it would be possible to replicate the findings reported by Fungwe et al., 1990 (the Fungwe study findings were not repeatable). The range-finder and the main study each included three dose levels via drinking water. Since for humans the exposure via the diet is the more relevant route of exposure, the range-finder also included 3 dose levels via the diet. For the main study, only the highest dose level was administered via the diet. The US EPA Office of Water was involved in the decision to select drinking water as the main route of administration in the main study. For technical reasons, four separate study records have been prepared in IUCLID for the drinking water and diet cohorts of the range-finder and main study, respectively. All parts of the study should be considered together.

 

No adverse effect on reproductive function was observed at any dose level in either generation as indicated by no significant dose-related effect on gonadal function, oestrus cycles, sperm parameters, mating, conception, gestation, parturition, lactation, litter size, pup survival, growth or postnatal development through to mating of the F1 and weaning of the F2 offspring. Serum levels of Mo and copper were increased in a dose-related manner. The No Observed Adverse Effect Levels (NOAEL) are 17 mg Mo/kg bw/day for systemic toxicity and 40 mg Mo/kg bw/day for reproductive toxicity. (see two separate study records “Hoberman, 2017” on the drinking water and dietary administration parts of this study.

Earlier, a guideline-compliant 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 monitoring 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 diet. 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 monitoring, sperm count, motility and morphology, and detailed testicular histopathological examination including 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/food conversion efficiency. 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 when evaluated during weeks 7-9 of the dosing phase. 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 female 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.

Other data:

The registrant, via the REACH Molybdenum Consortium has further conducted an extensive literature/data search and evaluation programme on animal and human toxicity data relating to reproductive/developmental toxicity of molybdenum substances. All data sources were assessed by expert toxicologists for quality and reliability, as well as relevance for regulatory risk assessment under REACH. Besides the recent guideline studies by Hoberman (2017), supported by Hoffmann et al. (2011), no further reliable data on fertility and reproductive performance was found. Further relevant but unreliable data is presented in the technical dossier and in tabular format in the CSR, but not further discussed in this endpoint summary. Reference is made to a tabular evaluation report (attachment in the technical IUCLID dossier, in the Endpoints Summary for section 7.8, under "Attached background material").

Effects on developmental toxicity

Description of key information

The conclusions on prenatal developmental toxicity are based on two studies, with three separate endpoint study records (one for Tyl, 2013, two for Hoberman: 2020 Range-Finder and 2021 main study):

Tyl (2013): This guideline-compliant dietary prenatal developmental toxicity study (acc. OECD 414, under GLP) in rats determined unbounded NOAELs for maternal and developmental toxicity, both at 40 mg Mo/kg bw/day (100 mg/kg bw/day of the test item sodium molybdate dihydrate, abbreviated “SMD”), which was the highest dose level investigated.

The Tyl (2013) study, in particular the dose-selection was the subject of an ECHA compliance check - see decision number: CCH-D-2114356486-40-01/F. The decision was appealed by the Lead Registrant Climax Molybdenum B.V., Netherlands, with support from the IMOA REACH Molybdenum Consortium (MoCon) (Appeal A-006-2017; Board of Appeal Decision of 11 December 2018). The outcome of the appeal was that the initial CCH decision was upheld.

MoCon therefore implemented a programme of additional studies (palatability, range-finder, gavage, followed by the definitive study) to provide the requested additional data at higher dose concentrations (see details in field "Additional information").

The new definitive OECD TG 414 study (Hoberman, 2021) was conducted as an adaptation of OECD TG 414 to extend the dose range of the Tyl, 2013 study - using the maximum tolerated dose for pregnant rats.

In conclusion, considering both the Tyl (2013) study and the Hoberman (2021) study, the following effect levels are established, expressed in terms of the test item sodium molybdate dihydrate ("SMD") and in terms of the element molybdenum (Mo):

NOAEL, maternal: 100 mg SMD/kg bw/day, corresponding to 40 mg Mo/kg bw/day (Tyl, 2013)

LOAEL, maternal: 200 mg SMD/kg bw/day, corresponding to 80 mg Mo/kg bw/day (Hoberman, 2021)

Unbounded NOAEL for developmental abnormalities and irreversible foetal effects: > 300 mg SMD/kg bw/day, corresponding to > 120 mg Mo/kg bw/day (the maximum tolerated dose and thus the highest dose tested) (Hoberman, 2021).

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-04-03 to 2020-05-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2018-06-25

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: kept at controlled room temperature

Species:

rat

Strain:

other: Crl:CD(SD) Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC, USA
- Age at arrival: 161 g to 245 g (at randomization: 175 g to 226 g)
- Weight at arrival: 60 -77 days
- Fasting period before study:

- Housing:
Gestation period: individually housed in solid-bottomed cages
Postpartum period: each dam with a litter was housed together in a nesting box.
Nesting material: Bed-o'Cobs®.
Enrichment: I-chews, stainless steel loft, and Crink’l-Nest™

- Diet (ad libitum): Purina Certified Rodent Chow® #5002 meal (PMI® Nutrition International)

- Water (ad libitum; chlorine was added to the processed water as a bacteriostat; processed water was expected to contain no more than 1.2 ppm chlorine at the time of analysis)

- Acclimation period: 1-4 days

No known contaminants were considered to be in the food, water, nesting material and animal enrichment items that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 66°F to 77°F (19°C to 25°C)
- Humidity: 30 % to 70 %
- Air changes: min. 10 changes/hour (positive airflow)
- Photoperiod (hrs dark / hrs light): 12/12 (± 30 minutes)

Route of administration:

other: oral, diet

Vehicle:

other: base diet

Details on exposure:

DIET PREPARATION
Test substance test diets (diet: Purina Certified Rodent Chow® #5002 meal (PMI® Nutrition International, Richmond, IN)) were prepared at appropriate concentrations to meet dose level requirements using a conversion factor of 2.5 to convert from sodium molybdate dihydrate to molybdenum content. The formulations were prepared as needed based on 28-day stability and stored at room temperature throughout the duration of use on study.

The control groups received the carrier control substance alone (Purina Certified Rodent Chow® #5002 meal (PMI® Nutrition International, Richmond, IN). The carrier control substance diet was used as needed based on assigned 42-day stability, stored at room temperature throughout the duration of use on study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test diet samples were collected for analysis, as follows:
1) Concentration: all groups (time schedule: first preparation)
2) Homogeneity: all dose groups (time schedule: first preparation)
The homogeneity results obtained from the top, middle and bottom for the dose group preparations were averaged and utilized as the concentration results.

Additionally, samples were analysed for concentrations of molybdenum, copper, and sulfur.

Method:
Analyses were performed by inductively coupled plasma - mass spectroscopy (ICP-MS) using a validated analytical procedure (Wahlen 2005)*.

Concentration and homogeneity analysis:
Duplicate (approx. 20 g) sets of top, middle, and bottom samples for the sampling time point were analysed. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% of theoretical concentration. Each individual sample concentration result was considered acceptable if it was within or equal to ± 20% of theoretical concentration. For homogeneity, the criteria for acceptability were a relative standard deviation (RSD) of concentrations of ≤ 5% for each group.

Stability analysis:
Stability analyses were performed previously and it was demonstrated that the test substance is stable in the carrier control substance when prepared and stored under the same conditions at concentrations bracketing those or lower than those used in the present study.

Results:
Diet analyses for achieved concentration averaged 9.8% and 16.1% above the target values for the 200 and 300 mg SMD/kg bw/day groups, respectively, based on an average of six samples from each batch of prepared diet.

The relative standard deviation (RSD), indicating the homogeneity of the diet, was 3.4% and 9.2% for the 200 and 300 mg SMD/kg bw/day groups, respectively. The homogeneity RSDs were based on the average of the concentration two top, three middle, and three bottom samples from each batch of prepared diet.

Some achieved concentration values that were higher than targets were not considered to have affected the outcome of the study as they were above, not below, the target value, indicating that the animals received at least the nominal dietary concentrations.

*Wahlen R. 2005: The Use of Collision/Reaction Cell ICP-MS for the Determination of Elements in Blood and Serum Samples http://www.spectroscopyonline.com/usecollisionreaction-cell-icp-ms-determination-elements-blood-and-serum-samples.

Details on mating procedure:

- Impregnation procedure: purchased time mated
Female rats were naturally bred at the Supplier, by breeder male rats of the same source and strain, before shipment to the Testing Facility. The day mating occurred was designated gestation day 0. The rats were shipped to the Testing Facility after mating to arrive on gestation days 2, 3, 4, or 5.

Duration of treatment / exposure:

gestation days 6 to 20

Frequency of treatment:

daily

Duration of test:

21 days (caesarian section animals) or approx. 62 days (littering animals)

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

caesarian section animals; equivalent to 80 mg Mo/kg bw/day (nominal) or 94.1 ± 11.89 mg Mo/kg bw/day (actually consumed; based on food consumption and body weight)

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

caesarian section animals; equivalent to 120 mg Mo/kg bw/day (nominal) or 125.7 ± 22.80 mg Mo/kg bw/day (actually consumed; based on food consumption and body weight)

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

littering animals; equivalent to 120 mg Mo/kg bw/day (nominal) or 128.5 ± 25.47 mg Mo/kg bw/day (actually consumed; based on food consumption and body weight)

No. of animals per sex per dose:

24 female rats

Control animals:

other: yes, base diet (control group each for caesarean section animals and littering group)

Details on study design:

RATIONALE FOR ROUTE AND DOSE SELECTION:
- Route selection rationale: the dietary route was selected because this was the route of exposure for the study (Tyl, 2013) that this study is designed to supplement, and as gavage administration of higher dose levels was not tolerated. In a 14-day repeat-dose gavage study, 4/6 females were found dead after 5 doses at 1000 mg/kg/day (the limit dose for OECD 414 studies) and at 600 mg/kg/day all animals were terminated after 7 doses owing to severe toxicity. At 300 mg/kg/day all animals survived to termination on Day15 but terminal body weight was 20% lower than controls and food intake was reduced by 40%.

- Dose selection rationale: the dose levels were selected based on a dietary range-finding study in the pregnant rat (please also refer to IUCLID section 7.8.2 Developmental toxicity / teratogenicity: s_RL1_Hoberman_2020_Dose-Range-Finder) in which sodium molybdate dihydrate doses of 300 and 400 (120 and 160 mg Mo)/kg bw/day resulted in statistically significant (p ≤ 0.05 to p ≤ 0.01) dose dependent reductions in mean body weight (13.8%, 22.8% on gestation day 20), body weight gains (41%, 68% from gestation days 6 to 20) and corrected terminal body weight (minus the gravid uterine weight, 16% and 23% reductions) as compared to the control group. Overall food consumption values for the entire administration period (gestation days 6 to 20) for the 300 and 400 mg/kg bw/day groups were 19% and 39% lower than the control group value, respectively. Based on the consumed food measured each day, the overall achieved dose for the 300 mg sodium molybdate dihydrate (120 mg Mo)/kg bw/day group was 122.0 ± 26.1 mg Mo/kg bw/day and for the 400 mg sodium molybdate dihydrate (160 mg Mo)/kg bw/day group was only marginally higher at 134.75 ± 34.2 mg Mo/kg bw/day, owing to reduced food intake.

As effects in the 400 mg sodium molybdate dihydrate (160 mg Mo)/kg bw/day group were considered more severe than those required by OECD 414 for pregnant females (Beyer 2011)*, and as achieved Mo intake at 400 mg sodium molybdate dihydrate/kg bw/day was only marginally higher, a high dose level of 300 mg sodium molybdate dihydrate (120 mg Mo)/kg bw/day was selected for this study. This level was 3-fold the high dose level of 100 mg sodium molybdate dihydrate (40 mg Mo)/kg/day used in the previous developmental toxicity study (Murray 2014)* and 200 mg sodium molybdate dihydrate (80 mg Mo)/kg bw/day (2-fold) was selected as the intermediate dose level.

ADDITONAL GROUPS:
(females allowed to litter and rear offspring to weaning)
Additionally to the female rats sacrificed on gestation day 21, 48 female rats were assigned to 2 littering groups (control group and high dose group) of 24 rats which were allowed deliver naturally and raise their young to weaning.

Consistent with the dosing period specified in OECD 414, animals assigned to the two littering groups had a test substance-free period from gestation day 21 through lactation day 21 (rats that delivered a litter) or gestation day 25 (rats that did not deliver a litter) where only the carrier control diet was provided. The offspring were not directly given the test substance formulations but may possibly have been exposed to the test substance in utero or via maternal milk during lactation. On day 21 post-partum, pups were killed and the carcasses processed for skeletal evaluation.

Reference:
Beyer BK, Chernoff N, Danielsson BR et al. 2011. ILSI/HESI Maternal Toxicity Workshop Summary: Maternal Toxicity and Its Impact on Study Design and Data Interpretation. Birth Defects Research (Part B) 92:36-51.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Cage side observations and time schedule:
Viability: at least twice daily

Clinical observations: on day of arrival, during the administration period, and on the day of scheduled euthanasia (dams allowed to litter were observed for maternal behaviour daily during the lactation period)

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: gestation day 0 (at the Supplier), on the day of arrival, daily during the administration period (gestation days 6 to 21), on laction days 0, 4, 7, 14, 17, and 21 for littering females, and on the day of scheduled euthanasia.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption: Yes it was recorded daily during the administration period (gestation days 6 to 21), and for littering females on lactation days 0, 4, 7, 14, 17, and 21.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes

Caesarian section animals:
On gestation day 21, female rats were euthanized by carbon dioxide asphyxiation. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed for each rat. The reproductive tract was dissected from the abdominal cavity. The uterus was opened and the contents were examined. The foetuses were removed from the uterus. Furthermore, the following organs of the dams were weighed at necropsy: thyroid gland, kidney, liver, and placenta. Paired organs were weighed together.

Representative samples of the following tissues from rats were collected and stored on dry ice: uterus with cervix (all nonpregnant animals only), thyroid gland , gross lesions/masses, kidney, liver, ovaries (all nonpregnant animals only), and placenta.

A table of random units was used to select one control group rat from the caesarian-section females from which all tissues examined at necropsy were retained, in order to provide control tissues for any possible future evaluations of gross lesions.

The medial lobe of the liver, left kidney, and the placentae were collected and analysed for molybdenum, copper, sulfur, and iron content. The remaining lobes of the liver and right kidney were retained in 10% neutral buffered formalin and processed for histopathological examination.

Histopathology was performed on thyroid, liver and kidney for all animals of the ceasearean section group.

Littering groups:
Rats that died before scheduled termination were examined for the cause of death or condition as soon as possible after the observation was made. A gross necropsy was performed; the rats were examined for gross lesions.

Females that did not deliver a litter were euthanized by carbon dioxide asphyxiation on gestation day 25 and examined. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed for each rat. The uterus was stained with 10% aq (v/v) ammonium sulfide solution and examined for implantation sites; the number of implantation sites and pregnancy status was recorded. Gross lesions were retained.

The dam with no surviving pups was euthanized by carbon dioxide after the last pup was found dead, missing (presumed cannibalized), or euthanized and examined. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed. The number of implantation sites was recorded. Gross lesions were retained.

On lactation day 0, after a minimum of 20 dams/group delivered, one dam and litter/group were euthanized and examined. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed for each rat. The number of implantation sites was recorded. Gross lesions were retained.

On lactation day 21, surviving female rats were euthanized by carbon dioxide and examined. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed for each rat. The number of implantation sites was recorded. Gross lesions were retained.

OBSERVATIONS OF LITTERING GROUPS:
- natural delivery observations: natural delivery observations and the number and vital status of the pups were recorded. Beginning on gestation day 17, the dams assigned to litter were observed at least twice daily.

Ovaries and uterine content:

NOTE: Applicable to caesarean section animals only

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (total weight placentas and foetuses)
- Total weight placenta/litter
- Number and distribution of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Uteri of apparently nonpregnant animals were stained with 10% aq (v/v) ammonium sulfide solution and examined for implantation sites.

Blood sampling:

BLOOD SAMPLING AND ANALYSIS - DAMS (caesarean section animals only)
- Plasma: No
- Serum: Yes, analysis of concentrations of molybdenum, copper, sulfur, and iron as well as for analysis of thyroid hormones (triiodothyronine (T3), thyroxine (T4) and thyroid stimulating hormone (TSH)).

Analysis of concentration of elements:
On gestation day 21, following euthanasia, blood samples (1.0 mL) were collected via the inferior vena cava from all female rats within two hours of the start of the light cycle on the day of euthanasia, and as soon as possible after euthanasia.

Blood samples were placed into serum separator tubes, allowed to clot for at least 30 minutes at ambient temperature then centrifuged for 10 minutes at 3500 rpm in a refrigerated centrifuge set to maintain 4°C. The resultant serum was separated, transferred to uniquely labeled clear polypropylene tubes, and frozen immediately over dry ice until stored in a freezer set to maintain -20°C. The serum samples were analysed for concentration of molybdenum, copper, sulfur, and iron.

Analysis of thyroid hormones:
On gestation day 21, blood samples (1.5 mL) were collected via the inferior vena cava following euthanasia and transferred into serum separator tubes from all female rats for analysis of T3, T4) and TSH. The animals were bled within two hours of the start of the light cycle on the day of euthanasia and as soon as possible after euthanasia.

Blood samples were allowed to clot for at least 20 minutes (did not exceed 1 hour) then centrifuged at room temperature at 3500 rpm for 15 minutes. The resultant serum was separated and divided into two aliquots with 250µL of serum for TSH and the remaining serum for T3 and T4, and frozen immediately on dry ice. Samples designated for TSH were stored in a freezer set to maintain -20ºC and samples designated for T3 and T4 were stored in a freezer set to maintain 70°C. Total serum T3, total serum T4, and TSH were measured in rat serum using a validated analytical method.

BLOOD SAMPLING AND ANALYSIS - LITTERS ONLY
- Plasma: No
- Serum: Yes, analysis of concentrations of molybdenum, copper, sulfur, and iron

On lactation Day 0, after a minimum of 20 dams/group had delivered, one litter/group was arbitrarily selected for blood collection as soon as possible after the dam had completed delivery. Blood samples (0.5 mL to 1.0 mL) were collected from the pups via decapitation and pooled by sex/litter. Furthermore, on lactation day 4, blood samples (0.1 mL to 1.05 mL) were collected from the available culled pups via cardiac puncture following euthanasia and pooled by sex/group. Lastly, on lactation day 21, blood samples (1.0 mL) were collected from pups via the vena cava following euthanasia to provide a 1 mL blood sample/sex/group. Samples were pooled by sex/group.

The blood samples were processed as described above for the dams and analysed for concentration of molybdenum, copper, sulfur, and iron.

Fetal examinations:

FOETAL OBSERVATIONS AFTER CAESEAREAN SECTION

- External examinations: Yes
Foetuses were examined for sex and for external abnormalities. Late resorptions and dead foetuses were examined for external abnormalities and sex to the extent possible.

- Soft tissue examinations: Yes, half of each litter
Foetuses were examined for visceral abnormalities by using a modification of the microdissection technique of Staples(1974)*. Each foetus was fixed in Bouin's solution and the heads were subsequently examined by free-hand sectioning.

- Skeletal examinations: Yes, half of each litter
Foetuses were examined for skeletal abnormalities after staining with alizarin red S.

- Anogenital distance of all rodent pups: anogenital distance measurement were taken using a calibrated stereomicroscope, micrometer and ruler after euthanasia. For male pups, the anogenital distance was measured from the cranial edge of the anus to the base of the anogenital aperture. For females, the anogenital distance was measured from the cranial edge of the anus to the base of the urinary aperture.

Further observations of the foetuses were made as follows:
- body weight of each foetus was recorded.
. uterine distribution of foetuses was recorded.
- placentae was examined for size, colour, and shape.
- number of live and dead foetuses were recorded.

OBSERVATIONS OF LITTERING GROUPS:
The number and vital status of the pups were recorded at delivery. Pups that were found dead during delivery or at the completion of littering (lactation day 0) were evaluated for vital status at birth. The lungs were removed and immersed in water. Pups with lungs that floated were identified as liveborn and to have died shortly after birth. furthermore, pups that died (Days 1 to 10 postpartum) before scheduled termination were examined for gross lesions and the cause of death as soon as possible after the observation was made.

On lactation day 0, after a minimum of 20 dams/group delivered, one litter of pups per group were euthanized by decapitation or intraperitoneal injection of sodium pentobarbital. The kidneys, liver and carcass were collected and frozen on dry ice until dispatch to MSU for analysis. The same procedure was conducted with the culled pups euthanized by intraperitoneal injection of sodium pentobarbital on lactation day 4.

On lactation day 21, surviving pups were euthanized by carbon dioxide asphyxiation. The kidneys and liver were collected and frozen on dry ice until dispatch to MSU for analysis. Carcasses were retained in 99% isopropyl alcohol for skeletal examinations after staining with alizarin red S.

- viability check: litters were observed for dead pups at least twice daily and the pups in each litter were counted at least once daily.

- general appearance: pups were observed for clinical observations daily

- body weight: pups were weighed on lactation days 0 (birth), 4, 7, 14, 18 and 21.

*References:
- Staples RE. Detection of visceral alterations in mammalian fetuses. Teratology 1974;9(3):A37-A38.

Statistics:

Please refer to the field "Any other information on materials and methods incl. tables" below.

Indices:

The following parental indices and litter calculations were included, where applicable:
- Pregnancy rate = (number of animals pregnant / number of animals paired) x 100
- Pre-implantation loss = ((number of corpora lutea - number of implants) / number of corpora lutea) x 100
- Post-implantation loss = ((number of implants - number of live foetuses) / number of implants) x 100
- Sex ratio (% males) = (number of male foetuses / total number of foetuses) x 100
- Litter % of foetuses with abnormalities = (number of foetuses in a litter with a given finding / number of foetuses in litter examined) x 100
- Gestation length = the gestation length was calculated from gestation day 0 to the day the first pup was observed.
- Female pregnancy index = number of pregnant females / number of females paired
- Gestation index: (number of animals with live offspring / number of animals pregnant) x 100
- Live birth index = (number of live newborn pups / number of newborn pups) x 100
- Viability index = (number of live pups on day 4 postpartum / number of live newborn pups) x100
- Lactation index = (number of live pups on day 21 postpartum / number of live pups on day 4 (postculling) postpartum) x 100
- Survival index (pup survival of 14 days) = (number of live pups on day 14 postpartum / number of live pups on day 4 (postculling) postpartum) x 100
- Survival index (pup survival of 7 days) = (number of live pups on day 7 postpartum / number of live pups on day 4 (postculling) postpartum) x 100
- Post-implantation loss/litter = (number of implants - total newborn pups) / number of implants) x 100

Historical control data:

Historical control data was provided by the laboratory for the following parameters:
- reproductive indices
- foetal external abnormalities
- foetal soft tissue abnormalities
- foetal skeletal abnormalities
- foetal ano-genital distance (1 study)
- foetal skeletal ossification site averages
- natural delivery and litter parameters
- thyroid hormones

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

1) Gestation period
Caesarean section animals:
- 300 mg/kg bw/day: suspected dehydration (based on skin turgor) was observed in 3/24 (13 %) rats on occasions between days 8 - 17 of gestation. Furthermore, erected fur and hunched posture were observed in 1/24 (4 %) and 2/24 (8 %) rats on gestation day 21, respectively. Loss of fur was recorded for 1/24 (4 %) rat on gestation day 14. Also thin fur was observed for 1/24 (4 %) rat on gestation days 15 to 21. Lastly, swollen muzzle was recorded for 1/24 (4 %) rat on gestation day 14 only.

Littering group:
- 300 mg/kg bw/day: suspected dehydration (based on skin turgor) was observed in 2/24 (8 %) rats on occasions between days 8 - 13 of gestation. Furthermore, erected fur and hunched posture were observed in 3/24 (13 %) and 1/24 (4 %) rats on gestation day 21, respectively. During lactation clinical observations considered related to test substance were limited to 2 females with hunched posture on lactation day 0, one of which also had erect fur, and a further female showing hunched posture on lactation days 0 to 9.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Littering groups:
300 mg/kg bw/day: one rat was found dead on the day of parturition. No clear cause of death could be determined, but the death was not considered related to test substance because: 1) it was a single event; 2) sporadic death during delivery is known to occur in this stain of rat; and 3) the dam had erect fur on gestation day 21 but no other adverse clinical observations, gained weight during the administration period (69 g vs group mean gain of 68 g for gestation day 6 - 20), and food consumption was comparable to other rats in of the same group. Necropsy revealed no gross lesions and confirmed that the female was pregnant with 9 implantation sites.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1) Gestation period:

- 200 mg/kg bw/day: reductions in mean body weight of 11.2% on gestation day 21 and in mean body weight gain over the administration period of 27.1% were statistically significant (p ≤0.01) and dose-proportionate.

- 300 mg/kg bw/day: exposure to test substance resulted in statistically significant (p ≤0.01) reductions of 20.7% and 21.9% in Group 3 (caesarian) and Group 5 (littering) in mean body weight on gestation day 21 and in mean body weight gain over the administration period of 49.8% adn 50.6%.

Corrected mean terminal body weights (body weight at gestation day 21 minus the total placental weight and total foetal weight) were statistically significantly (p ≤0.01) reduced by 12.4% and 23.7% in the 200 and 300 mg /kg bw/day groups, respectively, as compared to the control group of the caesarian animals.


2) Lactation period
- 300 mg/kg bw/day: mean body weights on lactation day 0 were reduced by 15.6% (p ≤0.01) compared to the control group of the littering animals. Some recovery was apparent as administration of the treated diet ceased on day 21 of gestation, and mean body weight gains during the lactation period were higher than the control group for each interval evaluated, with a significant (p ≤0.05) increase occurring for lactation days 4 to 7. However, mean body weight remained lower than control values throughout the lactation period (p ≤0.05 to p ≤0.01).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

1) Gestation period:
Caesarean section animals and littering group:
- 200 and 300 mg/kg bw/day: mean food consumption was reduced (p ≤0.05 or p ≤0.01) in a dose-dependent manner compared to the control at all tabulated intervals in the 300 mg/kg bw/day group, and all but gestation days 11-12 in the 200 mg/kg bw/day group.

Mean food consumption over the exposure period (gestation days 6 - 20) was 10.7%, 26.3%, and 23.9% lower than control values in the 200 (caesarean animals) and 300 (caesarean and littering animals) mg/kg bw/day groups, respectively.

2) Lactation period
- 300 mg/kg bw/day: mean food consumption for lactation days 0 to 4 was significantly reduced (7.8%, p ≤0.05) in the 300 mg/kg bw/day group but comparable to control group values thereafter.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Caesarean section animals:
- 200 and 300 mg/kg bw/day: the mean absolute liver weight and mean liver weight relative to terminal body weight were statistically significantly (p ≤ 0.01 or p ≤0.05) lower than controls in the 200 and 300 mg /kg bw/day groups, demonstrating a dose-response trend.

- 200 and 300 mg/kg bw/day: statistically significant (p ≤0.01) reduced weight of gravid uterine contents (total weight of placentas and foetuses in each litter) occurred in the 200 and 300 mg/kg bw/day groups compared to control.

- no treatment-related effects on kidney, thyroid or placental weights.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Caesearean section animals:
Abnormal consistency of pancreas was observed in 0, 2, and 17 rats in the control, 200 and 300 mg SMD/kg bw/day groups, and abnormal consistency of the cecum was observed in in 0, 2, and 15 rats in these groups, respectively.
These findings have been observed in previous studies with sodium molybdate dihydrate but their toxicological significance is unknown, although reduced food intake is known to adversely affect anatomical pathology in rats (Moriyama et al 2008). As no similar observations were made in the females killed on lactation day 21, however, they are considered reversible and of no toxicological concern.

Moriyama, Tomoyuki et al. “Effects of reduced food intake on toxicity study parameters in rats.” The Journal of toxicological sciences vol. 33,5 (2008): 537-47. doi:10.2131/jts.33.537

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Caesearean section animals:
1) Kidney:
Test substance-related microscopic changes were present in the kidney. Renal changes consisted of tubular regeneration, which increased in incidence and severity with dose (19/24 and 23/23 animals at 200 and 300 mg/kg bw/day, predominantly minimal at 200 and minimal to moderate at 300 mg/kg bw/day, compared to 4 control animals with minimal effects), and minimal mononuclear cell infiltration, which was more prevalent at 300 mg/kg bw/day (12/23, compared to 6/24 at 200 mg/kg bw/day and 4/24 in controls).

2) Liver
Test substance-related microscopic changes were present in the liver. Dosage-related incidences of minimal to mild hepatocellular hypertrophy and vacuolation were observed at 200 & 300 mg/kg bw/day. The incidence of minimal glycogen accumulation was not dosage-related and minimal/mild karyocytomegaly was observed in half of the females at 300 mg/kg bw/day only. Minimal necrosis, observed at a low incidence in all groups, may have been artifactual and the relationship to test article administration was interpreted as uncertain.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

CLINICAL SIGNS
1) Gestation period
Caesarean section animals:
- 0 and 200 mg/kg bw/day: no adverse clinical observations occurred.

Littering group:
- 0 mg/kg bw/day: no adverse clinical observations occurred.

2) Lactation period (littering group only)
- 0 mg/kg bw/day: no adverse clinical observations occurred. Only one dam showed abnormal maternal behaviour for one day only (not nesting, nursing or grooming pups).
- 0 and 300 mg/kg bw/day: maternal care, including cleaning of pups at birth, nursing, grooming of pups and normal nesting behavior, was evident in both groups with only one control dam abnormal for one day only (not nesting with pups, not nursing pups and not grooming pups).

MORTALITY:
Caesarean section animals:
- 0, 200 and 300 mg/kg bw/day: there was no mortality related to the test substance. All rats survived to scheduled euthanasia.

Littering group:
- 0 and 300 mg/kg bw/day: there was no mortality related to the test substance.

BODY WEIGHT AND WEIGHT CHANGES:
1) Gestation period
Caesarean section animals:
- 0 mg/kg bw/day: mean body weights and mean body weight gains for the two control groups were comparable at all intervals.

FOOD CONSUMPTION AND COMPOUND INTAKE:
1) Gestation period
Caesarean section animals and littering group:
0 mg/kg bw/day: mean food consumption did not differ significantly between the two control groups (caesarean and littering animals) for any interval evaluated except for a 9.9% reduction in the control group of the littering animals compared to the control group of the caesarean animals on one day (gestation days 12/13, (p ≤0.05).

Compound intake:
Based on the daily food measurements, the overall mg Mo/kg bw/day consumed dose for the 200 mg/kg bw/day group (equivalent to 80 mg Mo/kg bw/day) was 94.1 ± 11.89 mg Mo/kg bw/day and for the 300 mg/kg bw/day groups (equivalent to 120 mg Mo/kg bw/day) was 125.7 ± 22.80 mg Mo/kg bw/day for animals of the caesarean group or 128.5 ± 25.47 mg Mo/kg bw/day for animals of the littering group.

ENDOCRINE FINDINGS
Caesarean section animals:

1) Triiodothyronine (T3) and thyroxine (T4):
The T3 and T4 levels were comparable across the groups examined with no statistically significant differences occurring, although all group mean values were slightly lower than the historical control mean.

2) Thyroid stimulating hormone (TSH):
The mean TSH values for all groups were lower than the historical control range. Concentrations for the control animals ranged from 208 pg/mL to 4477 pg/mL, with a mean value of 985 pg/mL. TSH C oncentrations ranged from 208 pg/mL to 4475 pg/mL (mean: 1488 pg/mL) and 1155pg/mL to 8928 pg/mL (mean: 3063 pg/mL) and there were 51.1% and 211% increases in mean concentrations for the females administered 200 and 300 mg/kg bw/day, respectively, compared to the control. A stati stically significant increase in TSH concentrations in the 300 mg/kg bw/day group was not considered adverse as there were no changes in T3 or T4 levels or on thyroid weight, no microscopic correlates in the thyroid, and as values for the treated groups were more comparable to historical control values t han the concurrent control group.


GROSS PATHOLOGICAL FINDINGS
Caesarean section animals:

1) Kidney
- 0 mg/kg bw/day: dilatation of the renal pelvis of the kidney was noted in one control rat.

2) Thyroid gland
- 0, 200 and 300 mg/kg bw/day: no findings were observed at gross examination of thyroid glands.

3) Placenta
- 0, 200 and 300 mg/kg bw/day: no visible lesions were observed

Littering group:
- 0 and 300 mg/kg bw/day: no maternal gross lesions were noted at scheduled euthanasia on lactation day 21 or in the single 300 mg/kg bw/day dam that was found dead during parturition.

ORGAN WEIGHT FINDINGS INCLUDING ORGAN/BODY WEIGHT RATIOS
Caesarean section animals:

1) Kidney
- 200 and 300 mg/kg bw/day: the mean absolute kidney weights were lower in the 200 and 300 mg/kg bw/day groups, reaching statistical significance (p ≤0.01) in the 300 mg/kg bw/day dose group, but as the mean kidney weights relative to body weight were higher than controls and as individual kidney weights relative to terminal body weight in the 200 and 300 mg/kg bw/day dose groups were within the control range the differences were attributed to test substance-related differences in body weight.

2) Placenta
- 200 and 300 mg/kg bw/day: the mean absolute placenta weight per litter in the 200 and 300 mg/kg bw/day dose groups was lower than controls, reaching statistical significance (p ≤0.01) at 300 mg/kg bw/day and was also attributed to test substance-related reduction in maternal and foetal body weight as weights relative to body weight were not statistically significant.

3) Thyroid gland
- 0, 200 and 300 mg/kg bw/day: there was no effect on thyroid weight.


HISTOPATHOLOGICAL FINDINGS - NON-NEOPLASTIC
Caesarean section animals:

1) Thyroid gland
- 0, 200 and 300mg/kg bw/day: no test article-related microscopic findings were noted in the thyroid glands. Findings observed at microscopic examination of thyroid glands were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals and were considered unrelated to administration of sodium molybdate dihydrate.
Colloid areas of mild to moderate severity and moderate to marked follicular cell height were observed in all animals, including controls. The incidence of cysts was comparable amongst the groups (6-9/24, 6/22) and there was only one animal in each doseed group with thymic ectopia, compared to 3 in the control group.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

200 and 300 mg/kg bw/day: total placental weights per litter was reduced by 11 % at 200 mg/kg bw/day, and 24% at 300 mg/kg bw/day, compared to control, reaching statistical significance in the high dose group only (p ≤0.01), but as relative weights were not significantly different, this was attributed to SMD-related reductions in maternal body weight.

Details on maternal toxic effects:

NUMBER OF ABORTIONS
Caesarean section animals:
- 0, 200 and 300 mg/kg bw/day: no early deliveries were observed in the control group or any treatment group.

PRE- AND POST IMPLANTATION LOSS
Caesarean section animals:
- 0, 200 and 300 mg/kg bw/day: there was no effect on percent pre-implantation loss. A slight increase in the percent post-implantation loss was observed at 300 mg/kg bw/day with values slightly higher than the historical control range of the laboratory. These increases were not considered related to the test substance because of the lack of statistical significance and as no increase in post-implantation loss was observed in the littering group also exposed to 300 mg/kg bw/day, where the mean value was 28% lower than the littering control group.


Littering animals:
- 0 and 300 mg/kg bw/day: the mean post-implantation loss (number of pups per litter on Day 0 postpartum/number of implantation sites) of 7.28% in the 300 mg /kg bw/day group was 27.8% lower than the control value of 10.07% (not statistically significant).

DEAD FOETUSES
Caesarean section animals:
- No dead fetuses were observed in any group.

CHANGES IN NUMBER PREGNANT
Caesarean section animals:
- 0, 200 and 300 mg/kg bw/day: all rats (n = 24 rats/group) were pregnant, with the exception of a single rat in the control group.


Littering animals:
0 and 300 mg/kg bw/day: a total of 22 dams in the control group and 23 in the 300 mg/kg bw/day group were pregnant, with 22 dams in each group completing delivery. One female and litter in each group was terminated on day 0 post-partum to provide serum and tissue samples for analysis.

CHANGES IN PREGNANCY DURATION
Littering animals:
0 and 300 mg/kg bw/day: the gestation length (gestation day 0 until the delivery of the first pup) was comparable, at 22 ± 0.4 days in the control group and 21.9 ± 0.6 days in the 300 mg/kg bw/day dose group.

EARLY AND LATE RESORPTIONS
Caesarean section animals:
- 0, 200 and 300 mg/kg bw/day: an increase in the number of early resorptions per litter was observed at 300 mg/kg bw/day with the group mean comparable to the upper range of the historical control range of the laboratory. This increase was not considered related to the test substance because of the lack of statistical significance and as post-implantation loss at 300 mg/kg bw/day was lower than controls in the littering groups.


CORPORA LUTEA:
Caesarean section animals:
- 0, 200 and 300 mg/kg bw/d: there was no intergroup difference in the mean number of copora lutea per female


IMPLANTATIONS:
Caesarean section animals:
- 0, 200 and 300 mg/kg bw/d: there was no effect of the test substance on the mean number of implantations per female.

Littering group:
The mean number of implantation sites per female at 300 mg/kg/day was comparable to controls in the littering group.

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

100 mg/kg bw/day of the test item sodium molybdate dihydrate, abbreviated SMD, correspond to 40 mg element Mo/kg bw/day.

Basis for effect level:

other: No maternal toxicity based on all parameters in the OECD TG 414 study.

Remarks on result:

other: This NOAEL has been established in the previous Tyl (2013) study, in which 40 mg Mo/kgbw/day was the highest dose. This study (Hoberman, 2021) is an adaptation according to OECD TG 414 to extend the dose range of the Tyl study.

Key result

Dose descriptor:

LOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

200 mg/kg bw/day of the test item sodium molybdate dihydrate, abbreviated SMD, correspond to 80 mg element Mo/kg bw/day.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: see Remarks

Remarks:

Moderate, dose-dependent maternal toxicity was observed at 200 mg SMD/kg/bw/day (Group 2), as indicated by significant reductions in mean body weight (11.2% at DG21, p ≤0.01), in mean body weight gain over the administration period (27.1% from DG 6 to 20, p ≤0.01) and in corrected maternal body weight at DG21 (12.4%, p ≤0.01) compared to controls (Group 1). Maternal food intake over the administration period (DG 6-20) was also significantly reduced (p ≤0.01), at 10.7% lower than control (Group 1).

Key result

Dose descriptor:

other: The following dose level was established as the maximum tolerated dose for pregnant females and toxicity is more severe than would be expected for this study type.

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

300 mg/kg bw/day of the test item sodium molybdate dihydrate, abbreviated SMD, correspond to 120 mg element Mo/kg bw/day.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: see Remarks

Remarks:

Exposure to SMD at 300 mg SMD (120 mg Mo)/kg bw/day, which is the maximum tolerated dose for pregnant females, resulted in marked maternal toxicity, as indicated by statistically significant reductions in mean body weight of 20.7%, and 21.9% on DG 21 in Groups 3 (caesarian section animals) and 5 (littering animals), respectively, and reductions in mean body weight gain of 49.8%, and 50.6% over the administration period (from DG 6 to 20) compared to controls (Group 1).

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Caesarean section animals:
- 0, 200 and 300 mg/kg bw/day: reductions in male and female and combined foetal body weight observed at 200 and 300 mg/kg bw/day were statistically significant (p ≤0.01) and dose-proportionate.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

OFFSPRING VIABILITY:
Littering animals:
0 and 300 mg/kg bw/day: the viability and lactation indices (% of pups surviving
to Day 4 or from Day 4 to 21 postpartum, respectively) were comparable between the groups.

CHANGES IN SEX RATIO
Littering animals:
0 and 300 mg/kg bw/day: The litter sex ratio was comparable between the groups.

CHANGES IN LITTER SIZE AND WEIGHTS

Caesarian section animals:
There was no adverse effect on litter size. Litter weight was not recorded but foetal weights were reduced proportionate to maternal body effects.

Littering animals:
- 0 and 300 mg/kg bw/day: mean pup weights (combined sexes) in the 300 mg/kg bw/day group were significantly lower (p ≤0.01) than control at each interval measured (Days 0, 4-preculling, 4-postculling, 7, 14, 18, and 21 postpartum). Consistent with the reduced foetal weight observed at gestation day 21, mean pup weights were 19.2% lower at birth, but pup growth exceeded controls and mean weights were only 9.4% lower by Day 21 postpartum. Male and female pup weights were similarly affected, at 19.4 and 19.0% lower than controls at postnatal day 0, respectively, and 8.8% and 10.5% lower at Day 21 postpartum.


ANOGENITAL DISTANCE
Caesarean section animals:
- 0, 200 and 300 mg/kg bw/day: mean anogenital distance was significantly reduced (p ≤0.05 or p ≤0.01) in males, and for males and females combined, at 200 and 300 mg/kg bw/day compared to control. After adjustment for foetal body weight, a statistically significant (p ≤0.05) reduction occurred for males in the 300 mg/kg bw/day dose group only, but the difference from control was only 6% and standard deviations were high. The toxicological significance of this finding is questionable in the context of results from the dose range-finding study (please also refer to IUCLID section 7.8.2 Developmental toxicity / teratogenicity: s_RL1_Hoberman_2020_Dose-Range-Finder), where values for the two untreated control groups (10 litters/group) were significantly different, and both were lower than the control group in the current study and available historical control data (1 study), indicating considerable biological variation. Mean female anogenital distance and adjusted values were comparable across all groups in this study but also differed from previous study results.

When this degree of variation is considered, the small (6%) reduction in adjusted male foetal anogenital distance at 300 mg/kg/day was considered attributable to the marked reduction in foetal weight and marked maternal systemic toxicity.


EXTERNAL MALFORMATIONS
Caesarean section animals:
Fetal evaluations were based on 281, 291, and 275 live DG 21 Caesarean-delivered fetuses in the 0, 200 and 300 mg SMD/kg bw/day groups, respectively.

- 0, 200 and 300 mg/kg bw/day: there were no test item-related foetal external malformations or variations observed at any dose level. A total of two external anomalies occurred, and they were limited to the control foetuses. One foetus had a skin tag (variation, associated with mechanical damage) and the other foetus had a cranial meningocele (malformation). No other external anomalies occurred in any foetus.

VISCERAL MALFORMATIONS
Caesarean section animals:
134, 140 and 130 fetuses were examined for visceral abnormalities in the 0, 200 and 300 mg/kg bw/ day groups, respectively.
0, 200 and 300 mg/kg bw/day: there were no test item-related foetal visceral malformations or variations at any dose level. The control foetus with cranial meningocoele had a misshapen and small brain (malformation). One foetus in the 200 mg/kg bw/day group had situs inversus (malformation) and one foetus in the control had an absent innominate artery (variation). No other visceral anomalies occurred in any foetus.

These visceral malformations and variations were considered unrelated to the test substance because: 1) each observation was limited to 1 foetus at any dose level; 2) there was no dose relationship; 3) the observation was limited to the control group; and/or 4) the litter and/or foetal incidences were with in the historical control range for the laboratory.


SKELETAL MALFORMATIONS
Caesarean-derived foetuses:
147, 151, and 145 fetuses were examined for skeletal abnormalities and mean number of fetal ossification sites in the 0, 200 and 300 mg/kg bw/day groups, respectively.
- 0, 200 and 300 mg/kg bw/day: there were no test item-related foetal skeletal malformations or variations at any dose level. A single foetus in the 200 mg/kg bw/day group had sternoschisis (malformation). No other foetal skeletal malformations occurred in any foetus in any group.

All skeletal abnormalities were considered unrelated to the test item because: 1) the findings were limited to 1 to 3 litters in any group; 2) the findings were not dose-dependent; and/or 3) the litter and foetal incidences were within the historical range for the laboratory.

Mean foetal ossification sites:
Caesarean section animals:
- 0, 200 and 300 mg/kg bw/day: the mean number of ossification sites per foetus for the areas assessed, i.e. hyoid, vertebrae (cervical, thoracic, lumbar, sacral), rib pairs, and sternum (manubrium, sternal centers, and xiphoid) were unaffected by maternal administration of sodium molybdate dihydrate.

Reductions in mean foetal ossification sites occurred in those areas known to ossify in late gestation and to be transient findings (De Sesso & Scialli 2018)*, i.e. caudal vertebrae, phalanges (forelimbs and hindlimbs) and tarsals/metatarsals (hindlimb). The group incidences were dose dependent, reflecting the reduced mean foetal weights observed. The reductions were statistically significantly only in the 300 mg/kg bw/day dose group (p ≤ 0.05 or p ≤ 0.01) and values were generally within the historical control range.
Litters at Postnatal Day 21: Skeletal examination of all pups in the control and 300 mg/kgbw/day littering groups confirmed that all were fully ossified by Day 21 post-partum and it was concluded that any differences observed at gestation day 21 were transient.


CLINICAL SIGNS
Litter:
- 0 and 300 mg/kg bw/day: clinical observations for the pups in each litter were comparable between the groups.

GROSS PATHOLOGY
Litter:
- 0 and 300 mg/kg bw/day: at necropsy of dead pups, no milk in the stomach occurred in 5 pups from 2 litters in the control group and 1 pup in the 300 mg/kg bw/day group. No adverse effect of treatment was concluded.


*Reference:
- De Sesso JM and Scialli AR (2018): Bone development in laboratory mammals used in developmental toxicity studies. Birth Defects Research 110(15),1157-1187, DOI: 10.1002/bdr2.1350

Key result

Dose descriptor:

NOAEL

Effect level:

> 300 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

300 mg/kg bw/day of the test item sodium molybdate dihydrate, abbreviated SMD, correspond to 120 mg element Mo/kg bw/day. This was the maximum tolerated dose for pregnant female rats.

Sex:

male/female

Basis for effect level:

other: NOAEL for developmental abnormalities and irreversible foetal effects

Remarks on result:

other: see Remarks

Remarks:

There was no effect on the incidence of fetal external, visceral or skeletal malformations or variations. Fetal effects were limited to dose-dependent reductions in mean fetal weight (associated with marked maternal toxicity). The slight differences in ossification status observed in fetuses at DG 21 in the 300 mg SMD/kg/day group (120 mg Mo/kg bw/day) were confirmed as transient by skeletal examination of pups at Day 21 post-partum, and considered consistent with the reduced fetal weight, associated with marked maternal toxicity observed at this dose level. Pups delivered by females treated at 300 mg/kg bw/day were lower in weight than controls but pup growth to weaning exceeded controls.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

In conclusion, administration of sodium molybdate dihydrate in the diet at 200 or 300 mg/kg bw/day (80 or 120 mg Mo/kg bw/day) from Day 6 to 20 of gestation produced clear dose-dependent moderate to marked maternal toxicity, including adverse clinical observations, reductions in maternal weight gain (27.1% and 49.8% lower than control), and food intake (11% and 25%) over the administration period and reduced corrected (for uterine content) body weight at gestation Day 21 (12.4 and 23.7% lower than control). Liver weights were reduced and test item-related microscopic changes were present in the kidney and liver. Renal changes consisted of tubular regeneration and mononuclear cell infiltration, were more prevalent at 300 mg /kg bw/day. Liver changes included hepatocellular hypertrophy and glycogen accumulation at both dose levels and karyocytomegaly and vacuolation at 300 mg /kg bw/day. There was no adverse effect on thyroid weight, thyroid hormones or thyroid histopathology.

Foetal effects were limited to dose-dependent reductions in foetal weight (~11% and 22% at 200 and 300 mg/kgbw/day) but there was no effect on the incidence of external, visceral and skeletal foetal malformations and variations in the sodium molybdate dihydrate treated groups. The slight differences in the ossification status of foetuses in the 300 mg/kg bw/day group were confirmed as transient by skeletal examination of pups at Day 21 post-partum, and are consistent with the reduced foetal weight, associated with the marked maternal toxicity observed at this dose level.

Reference 2

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

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

This dossier is one of several dossiers prepared under the auspices of the REACH Molybdenum Consortium (“MoCon”). To avoid unnecessary (animal) testing, a comprehensive grouping and read-across concept has been developed amongst several molybdenum containing substances. This grouping/category approach is described in detail in a separate report, in accordance with the ECHA's "Read-Across Assessment Framework" (RAAF). This document is attached to section 13 in the technical dosser and to the CSR. The following text in this paragraph is therefore applicable to all substances in the category.

A guideline-compliant prenatal developmental toxicity study by Tyl (2013, 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. Therefore, the NOAEL for maternal toxicity was established at ca. 40 mg Mo/kg bw/day.

There were no biological or statistical differences among groups for the numbers of corpora lutea, implantation sites, or post implantation losses per female or litter size or sex ratios, foetal body weights, external, visceral or skeletal malformations or variations. 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  ca. 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.

This prenatal developmental toxicity (PNDT) study by Tyl (2013), in particular the dose-selection was the subject of an ECHA compliance check - see decision number: CCH-D-2114356486-40-01/F. The decision was appealed by the Lead Registrant Climax Molybdenum B.V., Netherlands, with support from the IMOA REACH Molybdenum Consortium (MoCon) (Appeal A-006-2017; Board of Appeal Decision of 11 December 2018). The outcome of the appeal was that the initial CCH decision was upheld.

MoCon then implemented a programme of additional studies to provide the requested additional data on intrinsic hazard for the PNDT data-requirement at higher dose concentrations.

This programme (2019-2021) included a 10-day dietary palatability study in non-pregnant females, a dietary range-finding developmental toxicity study in pregnant females and a 14- day gavage tolerability study in non-pregnant females, which collectively established the route providing the maximum tolerated dose for pregnant females in the prenatal developmental toxicity study.

The definitive study (Hoberman, 2021) was then conducted as an adaptation of OECD TG 414 to extend the dose range of the Tyl 2013 study at the maximum feasible dose of 120 mg Mo/kg bw/day (300 mg SMD/kg bw/day) and an intermediate dose of 80 mg Mo/kg bw/day (200 mg SMD/kg bw/day), i.e. 2 and 3-fold the previous highest dose of 40 mg Mo/kg bw/day (100 mg  SMD/kg bw/day). These dose levels elicited moderate to marked maternal toxicity, characterized by reductions of 27% (at 80 mg Mo/kgbw/day) and 50% (at 120 mg Mo/kgbw/day) in maternal weight gain. This degree of maternal toxicity observed at both dose levels far exceeds the 20% decrease in weight gain considered excessive by renowned experts (Beyer et al 2011)*. There was no adverse effect on pregnancy outcome and foetal effects were limited to a dosage-related reduction in foetal weight proportionate to maternal effects. Postnatal evaluations confirmed no adverse effect on pup growth to weaning. There was no adverse effect of treatment on the incidence of external, visceral or skeletal malformations or variations. Slight differences in ossification status at 120 mg Mo/kg bw/day were confirmed as transient by skeletal examination of pups at postnatal day 21 and were consistent with effects on foetal weight, consequent to the marked maternal toxicity observed at this dose level.

In conclusion, considering both the Tyl (2013) study and the Hoberman (2021) study, the following effect levels are established, expressed in terms of the test item sodium molybdate dihydrate ("SMD") and in terms of the element molybdenum (Mo):

NOAEL, maternal: 100 mg SMD/kg bw/day, corresponding to 40 mg Mo/kg bw/day (Tyl, 2013)

LOAEL, maternal: 200 mg SMD/kg bw/day, corresponding to 80 mg Mo/kg bw/day (Hoberman, 2021)

Unbounded NOAEL for developmental abnormalities and irreversible foetal effects: > 300 mg SMD/kg bw/day, corresponding to > 120 mg Mo/kg bw/day (the maximum tolerated dose and thus the highest dose tested) (Hoberman, 2021).

 

Other data:

The registrant, via the Molybdenum Consortium, has conducted an extensive literature/data search and evaluation programme on animal and human toxicity data relating to reproductive/developmental toxicity of molybdenum substances. All data sources were assessed by expert toxicologists for quality and reliability, as well as relevance for regulatory risk assessment under REACH. For enhanced IUCLID-readability only the key studies are discussed in this Endpoint Summary. Reference is made to a tabular evaluation report (attachment in the technical IUCLID dossier, in the Endpoints Summary for section 7.8, under "Attached background material").

 

Reference:

Beyer et al. (2011): ILSI/HESI maternal toxicity workshop summary: maternal toxicity and its impact on study design and data interpretation, Birth Defects Res B Dev Reprod Toxicol, 92(1):36-51. doi: 10.1002/bdrb.20281

Justification for classification or non-classification

The information available regarding the effects of molybdenum on fertility does not warrant hazard classification of this molybdenum substance for reproductive toxicity. The information available for developmental toxicity does not warrant hazard classification of this molybdenum substance for reproductive toxicity (Regulation (EC) No 1272/2008).

The outcome of the follow-up Prenatal Developmental Toxicity study (Hoberman, 2021, as an adaptation of OECD TG 414 to extend the dose range of Tyl, 2013 was limited to fetal toxicity (effects on foetal body weight) consequent to marked maternal toxicity, without adverse effect on development, i.e. no effect on the incidence of external, visceral or skeletal malformations and variations. The degree of maternal toxicity observed at both dose levels in the Hoberman (2021) study far exceeds the 20% decrease in weight gain considered excessive by renowned experts (Beyer et al 2011).

 

Beyer et al. (2011): ILSI/HESI maternal toxicity workshop summary: maternal toxicity and its impact on study design and data interpretation, Birth Defects Res B Dev Reprod Toxicol, 92(1):36-51. doi: 10.1002/bdrb.20281