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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
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:
- Physical apperance: white crystalline powder
- Stability: at least until February 2021
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

Test animals

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)

Administration / exposure

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)
Doses / concentrationsopen allclose all
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.

Examinations

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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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.

Maternal developmental toxicity

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.

Effect levels (maternal animals)

open allclose all
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).

Results (fetuses)

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

Effect levels (fetuses)

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.

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

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.