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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 Jun - 20 Dec 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Diniobium pentaoxide
EC Number:
215-213-6
EC Name:
Diniobium pentaoxide
Cas Number:
1313-96-8
Molecular formula:
Nb2O5
IUPAC Name:
diniobium(5+) pentaoxidandiide
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han) (Full Barrier)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: Females 11 - 12 weeks old on arrival at the test facility; Males 12 - 13 weeks at the start of pairing
- Weight at study initiation: Males 353 – 424 g (mean: 386.29 g, ± 20% = 309.03 – 463.55 g); Females 181 – 243 g (mean: 211.83 g, ± 20% = 169.47 – 254.20 g)
- Fasting period before study: No
- Housing: The animals were kept individually in ventilated cages (type III H, polysulphone cages) on Altromin saw fibre bedding (except during the pre-mating period when females were kept in groups of two animals and during the mating period when two females were paired with one male). The pregnant females were provided with nesting material towards the end of the pregnancy (e.g. at GD 18).
- Diet: Altromin 1324 maintenance diet for rats and mice (Altromin Spezialfutter GmbH & Co. KG, Lage, Germany), ad libitum.
- Water: tap water, sulphur acidified to a pH of approximately 2.8, ad libitum.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
aqua ad injectionem
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was ground into a fine powder and weighed into a tared plastic vial on a precision balance. Dose formulations were prepared by adding the required volume of vehicle (aqua ad injectionem) to obtain the appropriate final concentration of the test item.
Prepared formulations were vortexed and/or stirred until visual solubility was achieved.

VEHICLE
- Concentration in vehicle: 20 mg/mL (low dose; 100 mg/kg bw/day), 120 mg/mL (mid dose; 600 mg/kg bw/day) and 200 mg/mL (high dose; 1000 mg/kg bw/day)
- Amount of vehicle: 5 mL/kg bw
- Lot/batch no.: 911506 and 2005066
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verification of the test item formulations was conducted using ICP-OES. Before the beginning of the treatment period, fortified samples were prepared and analysed in order to obtain knowledge about recovery (70 - 110%) and stability of the test item in the selected vehicle at CIP Chemisches Institut Pforzheim GmbH as part of a separate GLP study (CIP study code 21B14017-01-VMWA, BSL Study No. 2100105).
Study pre-start stability analysis was included on the samples from high dose and low dose groups and the investigation was made for 0 h, 6 h (RT), 13 days (RT), and 13 days (2 - 8 °C). The test item formulations were shown to be stable for at least 13 days (both at RT and 2 - 8 °C).
Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups. Homogeneity samples were prepared by the test facility to guarantee comparability to test item formulations applied in the in vivo study. Exactly 1 mL of each homogeneity sample was sent to CIP Chemisches Institut for analysis (CIP study code: 21B14017-01-VMWA).
As the test item formulation was shown to be homogenous according to CIP study 21B14017-01-VMWA, samples were not collected during the study for the investigation of homogeneity and samples were only taken for substance concentration in the first and in the last week of the study for all doses (8 samples in total).
Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 1 mL). The A-samples were analysed at CIP Chemisches Institut Pforzheim GmbH (CIP study code: 21B14017-01-RAWA) and until then stored under appropriate conditions based on available stability data. The B-samples are retained at below 2 - 8 °C at BSL Munich (test facility) and discarded after completion of the final study report.
The determination of niobium contents in dose formulations was performed by ICP-OES using two independent wavelengths highly specific for niobium, one wavelength for quantification and one for confirmation. Recovery of test item concentrations were found between 82% and 114% of the nominal test item concentration in dose formulations.
Details on mating procedure:
- Impregnation procedure: Cohoused
- If cohoused: Mating was performed using a ratio of 1:2 (male to female). Females were paired for cohabitation in batches in order to control the number of animals for terminal sacrifice on a particular day. At the subsequent morning, the vaginal smear of the females was checked to confirm the pregnancy. The day on which sperm was observed in the vaginal smear was considered as Gestation Day (GD) 0. Mated females were assigned in an unbiased manner to the control and treatment groups ensuring that group mean body weights were comparable with each other. Each animal was assigned a unique identification number. After getting 92 sperm-positive females, the remaining females and males were discarded without any observation.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as GD 0.
Duration of treatment / exposure:
Day 5 - 19 of gestation
Frequency of treatment:
daily, 7 days/week

Inadvertently, dosing was missed on 08 July 2021 (GD 6 (animal no. 18, 19 and 41) and GD 16 (animal no. 16 - 17, 39 - 40, 63 - 64 and 86 - 87)) in a total of 11 female animals (C: 4 animals, LD: 3 animals, MD: 2 animals, HD: 2 animals). Therefore, additional females were included in the study and distributed to the respective groups. The animals were treated with the test item or vehicle on 7 days per week from GD 5 to GD 19.

Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day
Remarks:
Low dose (LD)
Dose / conc.:
600 mg/kg bw/day
Remarks:
Mid dose (MD)
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
High dose (HD)
No. of animals per sex per dose:
23 female Wistar rats per dose group

During the treatment period, inadvertently dosing was missed on 08 July 2021 in a total of 11 female
animals (control: 4 animals, LD: 3 animals, MD: 2 animals, HD: 2 animals). This was GD 6 for 2 control animals and 1 LD animal, and GD 16 for 2 animals in the control, LD, MD and HD groups, respectively.
It was therefore decided to mate additional females, which were distributed to the respective groups. Animals which did not receive dosing, were excluded during summary data preparation and evaluation was made with 22 Control, 23 LD, 21 MD and 22 HD group pregnant females.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence of dose levels were selected with a view to demonstrate any dose-related response and a NOAEL.
- Fasting period before blood sampling for (rat) dam thyroid hormones: not stated
- Time of day for (rat) dam blood sampling: no

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made at least once a day, preferably at the same time each day. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
- Cage side observations checked: Clinical observations were made for the following (not exclusive); spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed once before initiation of pairing to ensure that the body weights were within ± 20% variation. The sperm-positive females were weighed on GD 0, 5, 8, 11, 14, 17 and 20. Males were not weighed in this study except once before initiation of pairing.

FOOD CONSUMPTION: Yes
- Food consumption of sperm-positive females was determined for the following intervals: GD 0 - 5, 5 - 8, 8 - 11, 11 - 14, 14 - 17 and 17 - 20. On GD 20 food consumption was not determined for animals no. 1 - 3, 24 - 26, 47 - 49, 70 - 73. Food consumption was not measured for males during the entire study or for both males and females during the mating period.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Gestation Day 20
- Organs examined: Uterus with the cervix and thyroid/parathyroid.
Immediately after the termination or as soon as possible after death, the uteri were removed and the pregnancy status of the dams was confirmed. Uteri that appeared non-gravid were further examined by staining with 10% ammonium sulphide solution to confirm the non-pregnant status.
Thyroid/parathyroid glands from all dams were preserved in 4% neutral-buffered formaldehyde. The weight of thyroid/parathyroid glands was measured after 24 hours fixation. A histopathological evaluation was carried out on the preserved thyroid/parathyroid glands from all dams of all dose groups sacrificed at the end of the treatment period.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Blood sampling:
- Serum: Yes
- Other: Thyroid hormone levels from samples from all dams were assessed at the end of treatment prior to or as part of the sacrifice of the animals. At termination, blood samples were collected from the defined site in serum separator tubes and obtained serum was stored under appropriate conditions. Serum samples were assessed for thyroid hormone levels (T3, T4 and TSH) using ELISA.
Fetal examinations:
- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, half per litter
- Skeletal examinations: Yes, half per litter
- Head examinations: Yes, craniofacial examination of the heads of the fetuses used for the soft tissue examination of the first 20 litters per group.
- Anogenital distance of all live rodent pups: yes
Statistics:
A statistical assessment of the results of the body weight and food consumption was performed by comparing values of dosed animals with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, thyroid hormones and foetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There was no test item-related clinical signs observed in any of the treated groups. None of the females showed signs of abortion or premature delivery prior to the scheduled sacrifice.
Clinical signs observed occasionally during the treatment period of the study included hairless areas on the skin and fur for two control group animals and one high dose group animal. In addition, one control group animal showed right corneal opacity on GD 8 and one high dose group animal showed lacrimation in the left eye.
All these findings are considered to be incidental.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean body weight and mean body weight gain were increased with the progress of the study in the control and all the treated groups throughout the study period. No statistical significance was observed in both mean body weight and mean body weight gain. There was a slight increase or decrease in mean body weight gain observed during the treatment period when compared to control without statistical difference or consistency between the groups.
Overall, there were no test item-related effects on mean body weight or gain observed in the test item treated groups on GDs 5 - 20.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean food consumption observed during GDs 5 - 20 in the test item treated groups were comparable to control. No statistical significance was observed in mean food consumptions. No test item-related effect on mean food consumption was observed in the treated groups.
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:
not specified
Description (incidence and severity):
The following ED-related parameters were investigated in the study: T3, T4 and TSH levels in dams, anogenital distance, genital abnormalities (testicular descent/cryptorchidism), thyroid histopathology dams, thyroid weight dams, gravid uterus weight, litter size, litter/foetus weight, number of implantations, corpora lutea, number of embryonic or fetal deaths and viable foetuses, post-implantation loss, pre-implantation loss, presence of anomalies (external, visceral, skeletal) and sex ratio. For details, please refer to the respective result fields and the endpoint summary.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Statistical analysis of post-fixed thyroid/parathyroid weights from all dams revealed statistically significant differences in the absolute weight in the HD group but not in relative (to body weight) thyroid/parathyroid weights of the treated groups when compared to the control. There was a slight decrease in absolute mean thyroid/parathyroid weights (5.51%, 11.85% and 20.2% in LD, MD and HD groups) and relative mean thyroid/parathyroid weights (7.3%, 9.5% and 17.95% in LD, MD and HD groups) of the treated groups when compared to control. However, there were no macroscopic findings or changes in hormone levels or histopathological changes in the thyroid glands that could be related to the treatment with the test item.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test item-related gross pathological changes were observed during the macroscopic examination of any treatment groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No test item-related histopathological findings were observed in thyroid glands and parathyroid of all females.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
In all terminally sacrificed females, no statistically significant or toxicologically relevant effects were observed on group mean T3, T4 and TSH hormone levels and values were comparable to the control.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
None of the females showed any signs of abortion or premature delivery prior to their scheduled sacrifice.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No test item-related effects were observed on the percent of pre- and post-implantation loss.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No total litter loss by resorption was observed.
Early or late resorptions:
no effects observed
Description (incidence and severity):
No test item-related effects were observed on the percent of early and late resorptions.
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead fetuses were observed.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
No test item effects were observed on the number of animals pregnant.
Successful mating resulted in 26/26 pregnancies in the LD group, 23/25 in the MD group and 22/25 in the HD group compared to 26/27 pregnancies in the control group.
One female in control, 2 in MD and 3 in HD groups were found to be non-pregnant.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no test item related effects were observed for general maternal toxicity or maternal developmental toxicity at this dose level.

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean male and female foetal weights observed on an individual basis (sum of weight of all foetuses in the group divided by total number of foetuses in the respective group) in the LD, MD and HD groups were comparable to control. Statistically significant increase in HD group male foetus weight (3.6% above control) and in female foetus weight of MD and HD groups (5.74% and 3.27% above control, respectively) were observed. All these values were within the historical control range of this strain.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No test-item related effects were noted on the number of live foetuses in the treatment groups when compared to the control group.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No test-item related effects were noted in the number of male and female foetuses and sex ratios in the treatment groups when compared to the control group.
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant increase in mean female foetal weight was observed in MD group on a litter basis (group mean of individual litter mean) when compared to the control group. No other groups showed any statistical significance. Overall, there were no test item related effects on mean foetal weight on a litter basis in any of the test item treated groups.
Mean litter size in treated groups was comparable to the concurrent control.
Anogenital distance of all rodent fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
Statistical significance was observed in all treated groups for fetal AGD and relative AGD in female foetuses when compared to the control group. In male foetuses, statistical significance was observed in LD and HD groups for fetal AGD and relative AGD when compared to the control group. All these values were marginal and without any dose dependency. In addition, all these values were within the historical control range of this strain.
All male foetuses were checked for indications of incomplete testicular descent/ cryptorchidism and evaluation revealed the completion of testicular descent (abdominal) in all male foetuses from all groups).
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related external abnormalities were observed in any of the treated groups.
A single incidence of tail, kinked was observed in one foetus of the MD group.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal examination and examination of cartilage of the Alizarin red stained foetuses revealed a range of findings in all groups including control.
As usual for foetuses at this stage of gestation (Day 20), incomplete ossification was seen in several bones of litters in all treated groups and the control. Mostly, bones of the skull, sternum, paws, limbs and vertebra were affected by variations in the status of expected ossification, described in terms of incomplete ossification, irregular ossification, unossified or increased ossification throughout all experimental groups. However, there was no dose dependency and no trend towards a treatment related incomplete ossification of bones was observed. Statistical analysis did not reveal any significant changes when compared to control.
Bones showing a higher or lower litter incidence of incomplete ossification in the HD group compared to the control group which did not reach statistical significance were forelimb humerus (10% compared to 15%), scapulae (5% compared to 0%), skull mandibles (10% compared to 0%), skull parietal bone (75% compared to 70%), skull hyoid body (10% compared to 15%), skull basioccipital (10% compared to 5%), 4th sternebra (10% compared to 15%), skull squamosal (L) (15% compared to 5%), skull interparietal (90% compared to 100%), skull zygomatic arch (L) (15% compared to 25%), skull frontal (L) (10% compared to 5%), 2nd sternebra (50% compared to 55%), 5th sternebra (85% compared to 80%), skull supraoccipital (90% compared to 100%), hindlimb femur (35% compared to 30%), skull basisphenoid (25% compared to 50%), skull parietal (R) (10% compared to 30%), pelvic girdle pubis (15% compared to 5%), vertebra cervical arch (25% compared to 15%), vertebra thoracic arch(es) (10% compared to 0%), skull nasal (20% compared to 25%) and skull squamosal (B) (40% compared to 30%).
Bones showing higher or lower litter incidence of being unossified in the HD group compared to the control group, which did not achieve statistical significance were forelimb phalanx (95% compared to 100%), vertebra caudal arch (15% compared to 5%), vertebra cervical centrum (90% compared to 100%) and 6th sternebra (15% compared to 30%).
Bones showing a higher or lower litter incidence in the HD group compared to the control group which did not achieve statistical significance were bent scapula (25% compared to 15%), irregular ossification of vertebra thoracic centrum (25% compared to 30%), rudimentary 14th rib (65% compared to 60%), skull supraoccipital with small hole (60% compared to 45%), 14th rib-left rudimentary (50% compared to 40%), wavy ribs (75% compared to 80%), irregular ossification of vertebra cervical arch (30% compared to 40%) and 14th rib-right rudimentary (30% compared to 20%).
In general, wavy ribs are typically classified as transient and reversible post-natally and may thus be considered as variations but not malformations.
Observed ossification-related findings were observed at lower or higher incidences and without dose-dependency and thus were not considered as toxicologically relevant.
There were also slightly higher or lower litter incidences of skeletal findings in the LD and MD groups, but they did not show any statistical significance or dose dependency.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No statistical significance was observed in any of the treated groups when compared to the control group. Litter incidences of azygos vein (bilateral) were observed in the treated groups (5% in LD, MD groups and 0% in HD group, compared to 5% in control) when compared to the control.
Higher litter incidences for umbilical artery malpositioned were observed in the LD and MD group (60% and 65% respectively, compared to 50.0% in the control). Higher litter incidences of abdomen internal haemorrhage were observed (70%, 75% and 85% in the LD, MD and HD groups, respectively, compared to 65% in the control). Higher litter incidences of kidney with dilated renal pelvis was observed (5% and 10% in the LD and MD groups, respectively, compared to 0% in the control). Higher litter incidences of long thymus were observed (15% and 20% in the LD and HD groups, respectively, compared to 5% in the control).
Visceral findings observed in the treated groups were at frequencies generally comparable to or, in some cases, slightly higher or lower in frequency compared to the control. As the observed findings were either minor variations and/or due to a lack of dose dependency and consistency, no toxicological significance can be attributed to these findings and they were considered to be spontaneous in nature. In addition, all these values were within the historical control range for this strain.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Craniofacial examination by razor blade serial sectioning technique revealed a finding of midbrain, haematoma, subdural in LD group (15%) that was observed at slightly higher incidences when compared to control (5%). Slightly higher litter incidences of red material in the perimeningeal space of brain was observed in LD group (10%) when compared to control (5%).
These findings were considered to be spontaneous in nature and not related to the treatment with the test item. Statistical analysis of the data revealed no statistical significance for any of these findings. In addition, all values were within the historical control range for this strain.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed at this dose level.

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
In an oral gavage developmental study in rats, conducted in accordance to OECD 414 and in compliance with GLP, the NOAEL for diniobium pentaoxide for maternal toxicity and embryo/fetal development was ≥ 1000 mg/kg bw/day, as no adverse effects were observed up to the limit dose of 1000 mg/kg bw/day.