Molybdenum sulfide (MoS2), roasted

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Disodium molybdate

EC Number:

231-551-7

EC Name:

Disodium molybdate

Cas Number:

7631-95-0

Molecular formula:

Na2MoO4

IUPAC Name:

231-551-7

Test material form:

solid: particulate/powder

Details on test material:

solid, crystalline, white powder
purity > 99%
Molybdenum content in sample: 40 % (w/w)

Specific details on test material used for the study:

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

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The Sprague Dawley rat was chosen as the animal model for this study 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

Administration / exposure

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

Doses / concentrations

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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

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.

Effect levels (P0)

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

Results: P1 (second parental generation)

General toxicity (P1)

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 / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P1)

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.

Effect levels (P1)

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

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

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.

Effect levels (F1)

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

Results: F2 generation

General toxicity (F2)

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

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Details on results (F2)

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.

Effect levels (F2)

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

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

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.