Diniobium pentaoxide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Screening for reproductive / developmental toxicity (OECD 422): NOAEL ≥ 1000 mg/kg bw/day

Link to relevant study records

Reference

Reference 1

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity

Reproductive effects observed:

not specified

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 September 2009 to 12 April 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

(Urinalysis was not performed on the samples collected from five randomly selected males at the terminal sacrifice. Food was not withheld overnight prior to blood sampling. These deviations are considered to have no impact on the validity of the study.)

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System
Young healthy male and nulliparous, non pregnant female rats [strain: Wistar Crl:WI] (Full-Barrier), were used in this study. The animals were derived from a controlled full barrier maintained breeding system (SPF) (Source: Charles River, 97633 Sulzfeld, Germany).
According to Art. 9.2, No.7 of the German Act on Animal Welfare the animals were bred for experimental purposes.
At the beginning of the study, the age of the animals was 8-9 weeks. The range of the body weight was:
Females: 158.3-194.8 g, (mean: 176.82 g, ± 20%= 35.36 g)
Males: 236.1-275.4 g, (mean: 254.82 g, ± 20%= 50.96 g)

Housing and Feeding Conditions
After an adequate acclimatisation period (at least five days) the animals were barrier maintained (full-barrier) in air conditioned rooms under the following conditions:
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiol. controlled periodically)
- housed individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding
Certificates of food, water and bedding are filed at BSL BIOSERVICE.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Dosage
Based on the available information from other toxicity studies and in consultation with sponsor following doses were selected:
Control: 0 mg/kg bw/day
LD: 250 mg/kg bw/day
MD: 500 mg/kg bw/day
HD: 1000 mg/kg bw/day
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 is selected with a view to demonstrate any dosage related response and no observed adverse effect (NOAEL).
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle in the volume same as treated groups.

Administration of Doses
The animals were dosed with the test item on 7 days per week basis. The test substance was administered daily during 14 days pre mating and 14 days mating in both male and in female, during gestation period and up to post natal day 3 in females. Males were dosed for 28-29 days.
The test item was administered by gavage using a gavaging canula. The maximum dose volume administered was 10 mL/kg body weight.
For each animal the individual dosing volume was calculated on the basis of the most recently measured body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

All formulation samples were stored frozen (approximately -20°C) till the shipment to analytic laboratory and analysis is performed.
The dose formulation analysis was performed at CURRENTA GmbH & Co. OHG Services Analytik Building Q 25, 51368 Leverkusen, Germany.

Duration of treatment / exposure:

Males: 28-29 days.
Females: maximum 54 days

Frequency of treatment:

7 days per week

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes

Positive control:

no

Parental animals: Observations and examinations:

General clinical observations were made twice a day except during weekend and holidays where observations were made only once, approximately at the same time each day and considering the peak period of anticipated effects after dosing.Pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality were recorded.
Once before the first exposure, and once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoe, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), and piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypies, difficult or prolonged parturition or bizarre behaviour was recorded.
Sensory reactivity to different modalities, grip strength and motor activity assessments and other behaviour observations were conducted on five randomly selected males and females from each group. Observations were occurred during the last week of treatment in males and on day 3 of the lactation in females (only lactating females were evaluated).
The animals were weighed at randomisation, males weekly during the entire study period and at terminal sacrifice. Females were weighed weekly during pre mating period, on gestation day 0, 7/8, 14, 20 and on PND 1 (within 24 hours of parturition) and 4 along with pups.
Food consumption was measured on corresponding day of body weight after beginning of the dose administration. Food consumption was not measured during mating period.

Litter observations:

Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.

Postmortem examinations (parental animals):

All survived male animals were sacrificed on day 29 and 30. Non pregnant females were sacrificed on the respective day 26 after the sperm positive vaginal smear as an evidence of mating. Lactating females along with pups were sacrificed on respective post natal day 4.

Statistics:

Parameters like body weight change and food consumption was calculated for each animal as the difference in weight measured from one week to the next. The relative organ weights were calculated in relation to the body weight (measured at necropsy) and presented as percentage.
All results are reported in tabular form (summarized in mean or summary tables and listed in individual data tables). Mean body weights are also presented as figures.
Analytical results and histopathological findings are presented in separate phase-reports attached to this report.
For statistical analysis one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test was carried out to reveal any differences between control and test groups. Statistical analysis performed with GraphPad Prism V.x software (p<0.05 was considered as statistical significant).
In the evaluation of laboratory parameters, all values within a range of the mean value ± the two fold standard deviation (x ± 2s) are considered to be "normal“ values within a "normal“ population.

Reproductive indices:

calculated

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Clinical Observation and Mortality
No test item related clinical observations and mortalities were observed in male and
female animals during the entire period of the study. One female animal from low dose (18) was found dead on gestation day 15 due to gavaging error which was confirmed by microscopic evaluations and therefore not attributed to the treatment.

Detailed Clinical Observations/Functional Observation Battery
No relevant differences were observed concerning functional and behavioural examinations in male and females.
No abnormalities were recorded concerning posture, gait, palpebral closure, lacrimation, piloerection, arousal and vocalization.
No convulsions, tremors, stereotypy or bizarre behaviour were observed in animals of any groups.
For supported and unassisted rears, no abnormalities were detected. Responses to reflex testing were normal in all groups.
On measures such as counts of urination and defecation differences could not be detected.

Body Weight and Body Weight Change
In males, no effect on body weight and body weight change was observed through out the study period in treated groups when compared with controls.However, statistically significant decrease in overall body weight change during premating day 1 to terminal sacrifice was observed in mid dose group when compared with controls. Due to lack of dose dependency, this effect could be considered incidental and indicates no toxicological significance.
In females, statistical analysis of body weight and body weight change data revealed no difference during premating, gestation and lactation period except increase in body weight change during gestation period 0-7/8 in mid dose group as compared to controls. As increase was marginal and due to lack of dose dependency no toxicological relevance can be attributed to this finding.

Food consumption
In males, statistically significant decrease in food consumption during post mating period 1-7 was observed in low and mid dose group as compared to controls. However, lack of consistent and dose dependent pattern of effect on food consumption in treated groups indicates no toxicological relevance.
In females, No effect on food consumption was observed in treated groups during premating, gestation and lactation period as compared to controls.

Litter Weight Data
Statistical analysis of litter weight data revealed decrease in male litter weight on PND 0 in low and mid dose group and on PND 4 in low dose group. However, neither dose dependency nor an effect on group mean litter weight, total litter weight and female litter weight on PND 0 and PND 4 was observed when compared with controls. Therefore, this effect can not be considered as test item related.

Precoital Interval, Duration of gestation and fertility Index
No treatment related effect was observed on precoital interval, duration of gestation when compared with controls and values were comparable between the groups. All pregnancies resulted in normal births.
Successful mating resulted in 9, 10, 8 and 10 pregnancies in control, low mid and high dose respectively.
Reduced fertility index (No. of pregnant females/No. of copulated females X 100) was observed in control (90%) and mid dose group (80 %) as compared to low (100 %) and high dose group (100 %).

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

1 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: No adverse and treatment-related effects were observed

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

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

Group mean number of corpora lutea, No. of implantation sites, number of live pups born on PND 0, percent preimplantation loss and post implantation loss remained unaffected due to treatment when compared with controls.
No treatment related effect was observed on litter data such as total number of pups born, live pups, No.of female pups, still birth and runt on PND 0 and total No. of live pups and No. of female pups on PND 4.
No statistically significant effect on survival of the pups from PND 0 to PND 4 was observed in any treatment group when compared with controls.
One pup (Pup No. 1 male) from female No. 35 and two pups (pup no 4 male and 10 female) from female No 36 of high dose group were found dead on PND 1. Gross necropsy findings of found dead pup revealed no specific findings.

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: No adverse and treatment-related effects were observed.

Reproductive effects observed:

no

 

Reproductive Indices Index Group     C (0 mg/kg) LD (250 mg/kg) MD (500 mg/kg) HD (1000 mg/kg)   Copulation Index (%) 100 100 100 100 Fertility Index (%) 90 100 80 100 Delivery Index (%) 100 100 100 100 Viability Index Mean 100.00 100.00 100.00 97.00               Copulation Index (%)= (No. of rats copulated /No.of pairs)X 100 Fertility Index (%)= (No. of Females Pregnant/No.of females copulated)x 100 Delivery Index(%)= (No. of dams with live newborns/ No.of pregnant dams)X 100 Viability Index (%)= (No. of live offspring at day 4/ No.of live offspring at birth)x 100 No. of live offspring at day 4/ No.of live offspring at birth)x 100   Macroscopic Findings - Male Findings (External/Internal) C (0 g/kg) LD (250 mg/kg) MD (500 mg/kg) HD (1000 mg/kg) Total number of animals examined 10 10 10 10 Left seminal vesicle diminished 1 0 0 0 Epididymidis-yellowish, whitish deposition (bilateral) 1 0 1 0 Right epididymis-yellowish, whitish deposition 0 1 1 0 Lung redish discolouration 0 0 1 0 Left Epididymidis-yellowish, whitish deposition 0 0 1 1 Right seminal vesicle with white spots( 1-2mm) 0 0 0 1   Macroscopic Findings- Female Findings (External/Internal) C (0 g/kg) LD (250 mg/kg) MD (500 mg/kg) HD (1000 mg/kg) Total number of animals examined 10 10 10 10 Lymph nodes axillary: left slightly enlarged; right red discolouration at the margin 1 0 0 0 Lung-residuals of the test item 0 2 1 2 Lung-bloody infiltrated,foam 0 1 0 0 Kidney- small cyst on right kidney. 0 0 1 0 Thymus- slightly reduced in size 0 0 2 0 Lung-slight bloody, red spots 0 0 1 0 oesophagos-residuals of the test item 0 0 1 0 Axillary lymphnode-left slightly enlarged 0 0 0 1 Lung- bloody, red discoloured 0 0 0 2     Tabular Study Summary OBSERVATIONS Group Dosage (units). C (0 mg/kg) LD (250 mg/kg) MD (500 mg/kg) HD (1000 mg/kg) Pairs started (N) 10 10 10 10 Females showing evidence of copulation (N) 10 10 10 10 Females achieving pregnancy (N) 9 10 8 10 Conceiving days 1 - 5 (N) 9 10 10 10 Conceiving days 6 - . . .(1) (N) 1 0 0 0 Pregnancy = 21 days or below (N) 0 0 0 0 Pregnancy = 22 days (N) 6 5 7 7 Pregnancy ³ 23 days (N) 3 4 1 3 Dams with live young born (N) 9 9 8 10 Dams with live young at day 4 pp (N) 9 9 8 10 Corpora lutea/dam (mean) 12.56 12.67 12.88 12.30 Implants/dam (mean) 11.33 10.67 11.00 10.70 Live pups/dam at birth (mean) 10.89 10.11 10.63 10.20 Live pups/dam at day 4 (mean) 10.89 10.11 10.63 9.90 Sex ratio (m/f) at birth (mean) 1.78 0.85 0.98 1.16 Sex ratio (m/f) at day 4 (mean) 1.78 0.85 0.98 1.12 Litter weight at birth (mean) 65.10 62.70 62.64 59.61 Litter weight at day 4 (mean) 111.18 107.44 108.90 101.48 Pup weight at birth (mean) 6.02 6.26 5.91 5.87 Pup weight at day 4 (mean) 10.19 10.74 10.42 10.30 ABNORMAL PUPS Dams with 0 8 8 6 8 Dams with 1 1 0 2 1 Dams with 2 0 1 0 1 Dosage (units). C (0 mg/kg) LD (250 mg/kg) MD (500 mg/kg) HD (1000 mg/kg) LOSS OF OFFSPRING Pre-implantation (corpora lutea minus implantations) Females with 0 4 1 3 3 Females with 1 2 2 2 1 Females with 2 1 3 2 4 Females with 3 and more 2 3 1 2 Pre-natal (implantations minus live births) Females with 0 6 5 5 6 Females with 1 2 3 3 3 Females with 2 1 1 0 1 Females with 3 0 0 0 0 Post-natal (live births minus alive at post natal day 4) Females with 0 9 9 8 8 Females with 1 0 0 0 1 Females with 2 0 0 0 1 Females with 3 0 0 0 0

Conclusions:

In conclusion, the repeated dose administration of Diniobium Pentaoxide (Nb2O5) in deionised water to the male (28-29 days) and female (maximum 54 days) Wistar rats at dosages of 250, 500 and 1000 mg/kg bw/day revealed no major toxicological findings.
Based on the data generated from this combined repeated dose toxicity and reproduction/ developmental toxicity screening test with Diniobium Pentaoxide, the no observed adverse effect level (NOAEL) is believed to be 1000 mg/kg bw/day in males and females.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.7, of Regulation (EC) No 1907/2006.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Diniobium pentaoxide is insoluble in water. It is resistant to acids, including nitrohydrochloric acid (aqua regia), HCl, H2SO4, HNO3, and H3PO4 and to many organic and inorganic compounds. Diniobium pentaoxide is attacked by hot concentrated mineral acids, such as HF and HF/HNO3mixtures, but is resistant to fused alkali (Nowak & Ziolek,Chem. Rev. 1999,99, 3603-3624). I.e., diniobium pentaoxide is dissolved only under extremely oxidising conditions that are not compatible with administration to animals.

Due to its insolubility it can be assumed that diniobium pentaoxide even if applied as pure powder will not be absorbed in the stomach and intestinal tract. The negligible bioavailability after oral application allows the prediction that the NOAEL for toxicity after repeated oral exposure will be greater than 1000 mg/kg bw/day. This value was confirmed by the combined repeated dose toxicity and reproduction/ developmental toxicity screening test, as well as in the pre-natal developmental toxicity study.

 

Combined repeated dose oral toxicity study with the reproduction/developmental toxicity screening test in rat

In an oral gavage study conducted in accordance to OECD TG 422 (adopted in 1996) and in compliance with GLP, four groups comprising of 10 male and 10 female rats were dosed by oral gavage at 250, 500 and 1000 mg/kg bw/day. The test substance was administered daily during 14 days pre mating and 14 days mating in both males and females, during gestation period and up to post-natal day 3 in females. Males were dosed for 28 - 29 days.

There were no adverse effects in either sex for clinical observations, mortality, body weight, food consumption, functional and behavioural endpoints, haematology and clinical chemistry parameters, organ weights and gross or microscopic findings.

No reproductive or developmental toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day). No adverse effects were noted in any of the reproductive parameters (i.e. mating and fertility indices, precoital time, gestation length, number of implantations, spermatogenic profiling and histopathological examination of reproductive organs) and developmental parameters (i.e. sex ratio, viability indices, pup/litter weights and gross findings in offspring) investigated in the study.

In conclusion, based on the absence of adverse effects observed in the study, the NOAEL for reproductive and developmental toxicity was determined to be 1000 mg/kg bw/day.

 

Extended one-generation reproductive toxicity study: Annex IX waiver

According to Regulation (EC) No 1907/2006, Annex IX, 8.7.3, column 1, an extended one-generation reproductive toxicity study has to be performed  (basic test design (cohorts 1A and 1B without extension to include an F2 generation), one species) using the most appropriate route of exposure, if the 28-day or 90-day studies indicate adverse effects on reproductive organs or tissues or reveal other concerns in relation with reproductive toxicity. Data on subacute (28-day) oral toxicity (combined repeated dose toxicity and reproduction/developmental screening tests according to OECD 422) and subchronic (90-day) inhalation toxicity (OECD 413) conducted with the substance did not indicate any adverse effects on gross and histopathology of reproductive organs or tissues related to treatment in male and female rats. Furthermore, no effects on reproductive performance, including copulation rate and fertility as well as delivery and viability of pups, were observed after treatment in the OECD 422 study, nor were any effects on developmental toxicity observed in the OECD 414 study.

Effects on developmental toxicity

Description of key information

Pre-natal developmental toxicity (OECD 414): NOAEL (maternal and developmental toxicity) ≥ 1000 mg/kg bw/day

Link to relevant study records

Reference

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

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

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

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.

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

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.

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.

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.

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.

Key result

Abnormalities:

no effects observed

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.

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.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex IX, 8.7, of Regulation (EC) No 1907/2006.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Toxicity to reproduction: other studies

Additional information

Diniobium pentaoxide is insoluble in water. It is resistant to acids, including nitrohydrochloric acid (aqua regia), HCl, H2SO4, HNO3, and H3PO4 and to many organic and inorganic compounds. Diniobium pentaoxide is attacked by hot concentrated mineral acids, such as HF and HF/HNO3mixtures, but is resistant to fused alkali (Nowak & Ziolek,Chem. Rev. 1999,99, 3603-3624). I.e., diniobium pentaoxide is dissolved only under extremely oxidising conditions that are not compatible with administration to animals.

Due to its insolubility it can be assumed that diniobium pentaoxide even if applied as pure powder will not be absorbed in the stomach and intestinal tract. The negligible bioavailability after oral application allows the prediction that the NOAEL for toxicity after repeated oral exposure will be greater than 1000 mg/kg bw/day. This value was confirmed by the combined repeated dose toxicity and reproduction/ developmental toxicity screening test, as well as in the pre-natal developmental toxicity study.

 

Pre-natal developmental toxicity study in rats

In an oral gavage study conducted in accordance to OECD TG 414 and in compliance with GLP, pregnant female rats were exposed to diniobium pentaoxide during Gestation Days 5 to 19 at dose levels of 0, 100, 600 or 1000 mg/kg bw/day.

No test item-related mortality and no test item-related adverse clinical signs were observed in any of the treated animals during the study period.

No test item-related effects on mean body weight gain and food consumption were observed in any of the treated animals during the study.

No test item-related effects were observed on prenatal parameters including terminal body weight, adjusted maternal weight (carcass weight), uterine weight, number of corpora lutea, implantation sites, early and late resorptions, percent pre and post implantation loss, number of live foetuses, anogenital distance (AGD), foetal weight, number of male and female foetuses, sex ratios and testicular descent in treatment groups when compared to the control.

No test item related macroscopic and histopathological findings were observed in this study.

No statistically significant or toxicologically relevant effect was observed on group mean T3, T4 and TSH hormone levels. Statistical significance was observed in absolute thyroid/parathyroid weights at 1000 mg/kg bw/day, however, it was not considered to be test item related as there were no gross findings or changes in hormone levels, and no histopathological changes were observed in thyroid glands.

Furthermore, no test item-related and toxicologically relevant external, visceral, craniofacial and skeletal findings were observed in the treatment groups.

In conclusion, based on the absence of test-item related maternal and embryo/fetal effects in this study, the NOAEL for maternal toxicity and embryo/fetal development was ≥ 1000 mg/kg bw/day.

Justification for classification or non-classification

The available data is reliable and suitable for classification. Based on this data, classification for toxicity to reproduction according to Regulation (EC) No. 1272/2008 is not warranted.