Magnesium diniobate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-09-14 to 2009-10-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

repeated dose toxicity: oral, other

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-09-14 to 2009-10-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1996-03-22

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-04-06

Limit test:

no

Specific details on test material used for the study:

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

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Although several mammalian species may be used, the rat is the preferred rodent species.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: males: 236.1-275.4 g, (mean: 254.82 g); females: 158.3-194.8 g, (mean: 176.82 g)
- Housing: housed individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding
- Diet (ad libitum): Altromin 1324 maintenance diet for rats and mice
- Water (ad libitum): tap water, sulphur acidified to a pH of approx. 2.8
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10%
- Air changes: 10x/hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Details on route of administration:

No valid in vitro method is available for assessing combine repeated dose oral toxicity study with reproduction and developmental toxicity.

Vehicle:

other: deionised water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- the test item was suspended in the vehicle.
- the test item formulation was prepared freshly on each administration day before the administration procedure.
- dose volume: 10 mL/kg bw
- for each animal the individual dosing volume was calculated on the basis of the most recently measured body weight.

VEHICLE
- Justification for use and choice of vehicle: the vehicle was chosen due to its non-toxic characteristics as well as according to sponsor`s request.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each dosing concentration was analysed for nominal concentration by using ICP-MS. Stability and homogeneity of the test item in the vehicle was analyzed for the 250 and 1000 mg/kg bw/day dose concentrations.
Samples for the nominal concentration verification were taken in study week 1 (first week of pre mating period), 3 (first week of mating), 5 (gestation) and 7 (gestation/lactation).
Samples for homogeneity were taken from the top, middle and bottom of the 1000 mg/kg bw/day dose and 250 mg/kg bw/day dose preparation in study week 1 and 5.
Samples for stability analysis were taken in first week of the study. Small portion of preparations (in triplicate) was stored (approx. 24°C) for up to 14 days post-preparation to determine the stability of the test item in the vehicle.
All formulation samples were stored frozen until analysis.

Results:
1) Stability:
High dose: 0 hours: 91.5 % recovery; 6 hours: 58.0 % recovery
Low dose: 0 hours: 104.4 % recovery; 6 hours: 65.8 % recovery

2) Homogeneity:
High dose: week 1: 91.7 % to 98.7 % recovery; week 5: 68.4 % to 103.1 % recovery
Low dose: week 1: 81.8 % to 119.7 % recovery; week 5: 71.4 % to 106.8 % recovery

3) Concentrations (week 1, week 3, week5, and week 7):
High dose: 83.7 % to 98.8 % recovery (mean: 87.8 % recovery)
Mid dose: 86.3 % to 120.5 % recovery (mean: 97.6 % recovery)
Low dose: 87.5 % to 92.8 % recovery (mean: 90.3 % recovery)

Duration of treatment / exposure:

males: 28 - 29 days
females: approx. 53 days (14 days pre-mating, 14 days mating, during gestation period and up to post natal day 3)

Frequency of treatment:

daily

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 males / 10 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 is selected with a view to demonstrate any dosage related response and no observed adverse effect (NOAEL).

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day except during weekend and holidays where observations were made only once; observation approx. at the same time each day)
- Cage side observations checked: pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first exposure, and once a week thereafter

BODY WEIGHT: Yes
- Time schedule for examinations:
males: at randomisation, weekly during the entire study period and at terminal sacrifice
females: at randomisation, weekly during pre-mating period, on gestation day 0, 7/8, 14, 20 and on post natal day (PND) 1 (within 24 hours of parturition) and 4 along with pups

FOOD CONSUMPTION:
- Food consumption: Yes
Food consumption was measured on corresponding day of body weight after beginning of the dose administration. Food consumption was not measured during mating period.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No
OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at sacrifice
- Anaesthetic used for blood collection: Yes, Ketamin/Xylazin, 2:1
- Animals fasted: no
- How many animals: 5 males / 5 females from each group
- Parameters checked: haematocrit, haemoglobin, erythrocyte count, total leucocyte count, differential leucocyte count (banded neutrophils, segmented neutrophils, total neutrophils, basophil granulocytes, eosinophil granullocytes, lymphocytes, and monocytes), mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelet count, partial thromboplastin time, and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at sacrifice
- Animals fasted: no
- How many animals: 5 males / 5 females from each group
- Parameters checked: sodium, potassium, glucose, total cholesterol, urea, creatinine, total protein, albumin, alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the last week of treatment in males and on day 3 of the lactation in females (only lactating females were evaluated)
- Dose groups that were examined: 5 males / 5 females from each group
- Battery of functions tested: sensory activity / grip strength / motor activity / other behavioural observations

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- males were sacrificed after the completion of mating period (total dosing of 28-29 days)
- females were sacrificed on respective post-natal day 4 along with pups. Non pregnant females were sacrificed on the respective day 26 after the sperm positive vaginal smear as an evidence of mating.
- at the time of sacrifice, the adult animals were subjected to a full, detailed gross necropsy (included examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents). Special attention was paid to the organs of the reproductive system.

- number of implantation sites and corpora lutea was recorded for each lactating female at necropsy.

- ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicle with coagulating glands as a whole), and all organs showing macroscopic lesions of all adult animals were preserved and fixed.

Organ weights:
- reproductive organs from all animals were weighed (testes, epididymides, prostate, seminal vesicle with coagulating glands as whole, ovaries, uterus with cervix as applicable).
- from five males and females randomly selected from each group, the wet weight of the liver, kidneys, adrenals, thymus, spleen, brain and heart were taken as soon as possible. Paired organs were weighed separately and no organ weights were taken for found dead animals.

- the following tissues of same selected animals were preserved: all gross lesions, brain (incl. cerebrum, cerebellum and pons), spinal cord, liver, kidneys, adrenals, stomach, small and large intestines (incl. Peyer´s patches), thymus, thyroid, spleen, trachea, lungs, heart, urinary bladder, lymphnodes (mandibular and mesenteric), peripheral nerve (e.g. N. ischiadicus/ N. tibialis) in close proximity to muscle, and section of bone marrow

HISTOPATHOLOGY: Yes
- full histopathology was carried out on the preserved organs and tissues of the five randomly selected animals (male and female) in the control and high dose groups.
- testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) and all organs showing gross lesions were examined in all animals.
- for testis, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle.

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).
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 (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.

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- one female animal from 250 mg/kg bw/day dose group was found dead on gestation day 15 due to gavaging error which was confirmed by microscopic evaluations and therefore not attributed to the treatment.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

CLINICAL SIGNS:
- no test item related clinical observations were observed in male and female animals during the entire period of the study.

DETAILED CLINICAL OBSERVATIONS
- no relevant differences were observed concerning 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.

MORTALITY:
- no test item related mortalities were observed in male and female animals during the entire period of the study.

BODY WEIGHT AND WEIGHT CHANGES:
1) Males
- no effect on body weight and body weight change was observed throughout the study period in treated groups when compared with controls.
- statistically significant decrease in overall body weight change during premating day 1 to terminal sacrifice was observed in the 500 mg/kg bw/day dose group when compared with controls (lack of dose dependency; effect could be considered incidental and indicates no toxicological significance).

2) 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 the 500 mg/kg bw/day dose group as compared to controls (increase was marginal; lack of dose dependency; no toxicological relevance can be attributed to this finding).

FOOD CONSUMPTION AND COMPOUND INTAKE:
1) Males
- statistically significant decrease in food consumption during post mating period 1-7 was observed in the 250 and 500 mg/kg bw/day dose groups as compared to controls (lack of consistent and dose dependent pattern of effect on food consumption in treated groups indicates no toxicological relevance).

2) Females
- no effect on food consumption was observed in treated groups during premating, gestation and lactation period as compared to controls.

HAEMATOLOGICAL FINDINGS:
- in male and female, no statistically significant effect was observed in any of the hematological parameters of treatment groups except statistically significant increase in red blood corpuscles and haematocrit in 250 mg/kg bw/day dose group when compared with controls.

- individual values for few haematological parameters in few animals were below or above the biological range as follows:
white blood corpuscules: in two females from control group, individual values were below the biological range (3.3-21.5 x 10³/μL)
red blood corpuscles: in one male from the 250 mg/kg bw/day dose group, individual value was slightly above the biological range (3.7-9.57 x 10^6/μL.
haematocrit: in one male from 250 mg/kg bw/day dose group, individual value was above the biological range (32-53.7 %).
mean corpuscular volume: in one female from 1000 mg/kg bw/day dose group, individual value was above the biological range (47.4-62 fL)
mean corpuscular haemoglobin concentration: in one male from 250 mg/kg bw/day dose group, individual value was below the biological range (30.8-37.7 g/dL).
- almost all individual and group mean values for total neutrophils in all treatment groups and control group were below the biological range (5-57 %).
- the majority of individual and mean partial prothrombin time values (29-51 sec) and activated partial prothrombin time values (15-32.2 sec) were below the biological range in males. In females only activated partial prothrombin time individual and mean values were below the biological range.

- absence of consistent and dose dependent pattern of effect on various haematological parameters indicates no toxicological relevance.

CLINICAL BIOCHEMISTRY FINDINGS:
- statistical analysis of clinical chemistry data revealed decrease in aspartat-aminotransferase in 1000 mg/kg bw/day dose group male and females (no toxicological significance and can not be attributed to the treatment).
- all other clinical chemistry parameters remained unaffected due the treatment with test item and were within the biological range.
- individual values and group mean values for glucose, cholesterol and total protein were below or above the biological range as follows:
glucose: in 3 control, 4 low and all 5 mid and high dose male animals, individual and mean values were above the biological range (1.7-15.3 mmol/L).
cholesterol: in 2 control, 3 low, 1 mid and 3 high dose group males, individual and mean value were below the biological range (1.02-14.6 mmol/L). In one female from each group, individual values were below the biological range. (1.02 - 14.6 mmol/L).
total protein: in 1 control, 2 mid and 4 high dose group males, individual values were below the biological range (52-80 g/L). In 1 control, 2 low, 2 mid and 1 high dose females, individual values were below the biological range (52-80 g/L).

BEHAVIOUR (FUNCTIONAL FINDINGS)
- no relevant differences were observed concerning functional examinations in male and females.
- 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.

ORGAN WEIGHTS
1) Males
- no statistically significant difference in the absolute and relative organ weights of the treatment groups were found except a decrease in relative heart weights in 250 mg/kg bw/day dose group when compared with the controls (considered to be incidental due to lack of dose dependency).
2) Females
- statistically significant increase in absolute and relative thymus weights was observed in 250 mg/kg bw/day dose group when compared with controls (considered to be incidental due to lack of dose dependency).

GROSS PATHOLOGICAL FINDINGS:
1) Males
- various macroscopic findings observed were left seminal vesicle diminished (one male; control), yellowish whitish deposition on epididymides (control group: one male; 250 mg/kg bw/day dose group: one male; 500 mg/kg bw/day: three males; 1000 mg/kg bw/day: one male), redish discolouration of lung (500 mg/kg bw/day: one male) and white spot on right seminal vesicle (1000 mg/kg bw/day: one male).
2) Females
- various macroscopic findings observed were slightly enlarged and discoloured left axillary lymph node (control: one female; 1000 mg/kg bw/day: one female), lungs with remains of the test item (250 mg/kg bw/day: three females; 500 mg/kg bw/day: two females; 1000 mg/kg bw/day: two females), small cyst on right kidney (500 mg/kg bw/day: one female), slightly reduced thymus (500 mg/kg bw/day: two females) and bloody, discoloured lung (500 mg/kg bw/day: one female; 1000 mg/kg bw/day: two females).
- all other males and females from various treatment groups did not show any macroscopic findings at necropsy.
- gross pathological observation of male and females at scheduled necropsy revealed no treatment related findings and therefore observed findings are considered to be spontaneous in nature.

HISTOPATHOLOGICAL FINDINGS - NON-NEOPLASTIC
- in the female and male reproductive organs, as well as in the other organs evaluated in this study, no histopathological lesions were found which were considered to be test item related.
- three females from the study (control: one female; 250 mg/kg bw/day: two females) were non-pregnant and showed histopathologically metestrus/diestrus cycle in the sexual organs.
- in the lung, blackish intra-alveolar material, together with foci of pneumonitis or diffuse pneumonia, was noted in a low number of animals, mostly females, and was considered to represent misgavaged or aspirated test item.

Key result

Dose descriptor:

NOAEL

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

not specified

Conclusions:

After oral administration of 250, 500, and 1000 mg/kg bw/day of diniobium pentaoxide no adverse test item related effects were observed for clinical signs, mortality, body weight, body weight changes, food consumption, haematology, clinical chemistry, neurobehavioural examinations, organ weights, gross pathology, and histopathology.

In conclusion, the No Observed Adverse Effect Level (NOAEL) for the substance was concluded to be ≥ 1000 mg/kg bw/day for male and female rats.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1996-03-22

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-04-06

Limit test:

no

Justification for study design:

not applicable

Test material

Test material information

Constituent 1

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 pentaoxide

Test material form:

solid

Details on test material:

- State of aggregation: pale yellow solid
- Density: ~4.5 g/cm³

Specific details on test material used for the study:

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

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Although several mammalian species may be used, the rat is the preferred rodent species.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: males: 236.1-275.4 g, (mean: 254.82 g); females: 158.3-194.8 g, (mean: 176.82 g)
- Housing: housed individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding
- Diet (ad libitum): Altromin 1324 maintenance diet for rats and mice
- Water (ad libitum): tap water, sulphur acidified to a pH of approx. 2.8
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10%
- Air changes: 10x/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: deionised water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- the test item was suspended in the vehicle.
- the test item formulation was prepared freshly on each administration day before the administration procedure.
- dose volume: 10 mL/kg bw
- for each animal the individual dosing volume was calculated on the basis of the most recently measured body weight.

VEHICLE
- Justification for use and choice of vehicle: the vehicle was chosen due to its non-toxic characteristics as well as according to sponsor`s request.

Details on mating procedure:

- M/F ratio per cage: 1 male / 1 female
- Length of cohabitation: maximum 14 days (each morning after pairing the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated)
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of gestation

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each dosing concentration was analysed for nominal concentration by using ICP-MS. Stability and homogeneity of the test item in the vehicle was analyzed for the 250 and 1000 mg/kg bw/day dose concentrations.
Samples for the nominal concentration verification were taken in study week 1 (first week of pre mating period), 3 (first week of mating), 5 (gestation) and 7 (gestation/lactation).
Samples for homogeneity were taken from the top, middle and bottom of the 1000 mg/kg bw/day dose and 250 mg/kg bw/day dose preparation in study week 1 and 5.
Samples for stability analysis were taken in first week of the study. Small portion of preparations (in triplicate) was stored (approx. 24°C) for up to 14 days post-preparation to determine the stability of the test item in the vehicle.
All formulation samples were stored frozen until analysis.

Results:
1) Stability:
High dose: 0 hours: 91.5 % recovery; 6 hours: 58.0 % recovery
Low dose: 0 hours: 104.4 % recovery; 6 hours: 65.8 % recovery

2) Homogeneity:
High dose: week 1: 91.7 % to 98.7 % recovery; week 5: 68.4 % to 103.1 % recovery
Low dose: week 1: 81.8 % to 119.7 % recovery; week 5: 71.4 % to 106.8 % recovery

3) Concentrations (week 1, week 3, week5, and week 7):
High dose: 83.7 % to 98.8 % recovery (mean: 87.8 % recovery)
Mid dose: 86.3 % to 120.5 % recovery (mean: 97.6 % recovery)
Low dose: 87.5 % to 92.8 % recovery (mean: 90.3 % recovery)

Duration of treatment / exposure:

males: 28 - 29 days
females: approx. 53 days (14 days pre-mating, 14 days mating, during gestation period and up to post natal day 3)

Frequency of treatment:

daily

Details on study schedule:

not applicable

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 males / 10 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 is selected with a view to demonstrate any dosage related response and no observed adverse effect (NOAEL).

Positive control:

none

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day except during weekend and holidays where observations were made only once; observation approx. at the same time each day)
- Cage side observations checked: pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first exposure, and once a week thereafter

BODY WEIGHT: Yes
- Time schedule for examinations:
males: at randomisation, weekly during the entire study period and at terminal sacrifice
females: at randomisation, weekly during pre-mating period, on gestation day 0, 7/8, 14, 20 and on post natal day (PND) 1 (within 24 hours of parturition) and 4 along with pups

FOOD CONSUMPTION:
- Food consumption: Yes
Food consumption was measured on corresponding day of body weight after beginning of the dose administration. Food consumption was not measured during mating period.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No
OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at sacrifice
- Anaesthetic used for blood collection: Yes, Ketamin/Xylazin, 2:1
- Animals fasted: no
- How many animals: 5 males / 5 females from each group
- Parameters checked: haematocrit, haemoglobin, erythrocyte count, total leucocyte count, differential leucocyte count (banded neutrophils, segmented neutrophils, total neutrophils, basophil granulocytes, eosinophil granullocytes, lymphocytes, and monocytes), mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelet count, partial thromboplastin time, and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at sacrifice
- Animals fasted: no
- How many animals: 5 males / 5 females from each group
- Parameters checked: sodium, potassium, glucose, total cholesterol, urea, creatinine, total protein, albumin, alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the last week of treatment in males and on day 3 of the lactation in females (only lactating females were evaluated)
- Dose groups that were examined: 5 males / 5 females from each group
- Battery of functions tested: sensory activity / grip strength / motor activity / other behavioural observations

IMMUNOLOGY: No

REPRODUCTIVE PERFORMANCE:
- precoital interval, duration of gestation, births, and successful matings were recorded.
- signs of difficult or prolonged parturition were recorded.

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

Parameters examined in P male generations:
- testis weight and epididymis weight
- for testis, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined/recorded in F1 offspring as soon as possible after delivery of the dam:
- number and sex of pups
- stillbirths
- live births
- runts
- presence of gross anomalies
- weight within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum
- behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for gross abnormalities.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: after the completion of mating period (total dosing of 28-29 days)
- Maternal animals: on respective post-natal day 4 along with pups; non pregnant females were sacrificed on the respective day 26 after the sperm positive vaginal smear as an evidence of mating.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Special attention was paid to the organs of the reproductive system.

- number of implantation sites and corpora lutea was recorded for each lactating female at necropsy.

- ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicle with coagulating glands as a whole), and all organs showing macroscopic lesions of all adult animals were preserved and fixed.

HISTOPATHOLOGY / ORGAN WEIGHTS
- reproductive organs from all animals were weighed (testes, epididymides, prostate, seminal vesicle with coagulating glands as whole, ovaries, uterus with cervix as applicable).
- from five males and females randomly selected from each group, the wet weight of the liver, kidneys, adrenals, thymus, spleen, brain and heart were taken as soon as possible. Paired organs were weighed separately and no organ weights were taken for found dead animals.

- the following tissues of same selected animals were preserved: all gross lesions, brain (incl. cerebrum, cerebellum and pons), spinal cord, liver, kidneys, adrenals, stomach, small and large intestines (incl. Peyer´s patches), thymus, thyroid, spleen, trachea, lungs, heart, urinary bladder, lymphnodes (mandibular and mesenteric), peripheral nerve (e.g. N. ischiadicus/ N. tibialis) in close proximity to muscle, and section of bone marrow

HISTOPATHOLOGY: Yes
- full histopathology was carried out on the preserved organs and tissues of the five randomly selected animals (male and female) in the control and high dose groups.
- testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) and all organs showing gross lesions were examined in all animals.

Postmortem examinations (offspring):

SACRIFICE
- pups were sacrificed at day 4 post-partum, or shortly thereafter

GROSS NECROPSY
- Gross necropsy consisted of external examinations.

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).
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 (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:

Fertility index (%) = number of pregnant females / number of copulated females) x 100
Copulation Index (%)= (No. of rats copulated /No.of pairs) X 100
Delivery Index(%)= (No. of dams with live newborns/ No.of pregnant dams)X 100
Preimplantation loss (%)*
Post implantation loss (%)*
* no further information on calculation

Offspring viability indices:

Viability Index (%)= (No. of live offspring at day 4/ No.of live offspring at birth)x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- one female animal from 250 mg/kg bw/day dose group was found dead on gestation day 15 due to gavaging error which was confirmed by microscopic evaluations and therefore not attributed to the treatment.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS:
- no test item related clinical observations were observed in male and female animals during the entire period of the study.

DETAILED CLINICAL OBSERVATIONS
- no relevant differences were observed concerning 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.

MORTALITY:
- no test item related mortalities were observed in male and female animals during the entire period of the study.

BODY WEIGHT AND WEIGHT CHANGES:
1) Males
- no effect on body weight and body weight change was observed throughout the study period in treated groups when compared with controls.
- statistically significant decrease in overall body weight change during premating day 1 to terminal sacrifice was observed in the 500 mg/kg bw/day dose group when compared with controls (lack of dose dependency; effect could be considered incidental and indicates no toxicological significance).

2) 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 the 500 mg/kg bw/day dose group as compared to controls (increase was marginal; lack of dose dependency; no toxicological relevance can be attributed to this finding).

FOOD CONSUMPTION AND COMPOUND INTAKE:
1) Males
- statistically significant decrease in food consumption during post mating period 1-7 was observed in the 250 and 500 mg/kg bw/day dose groups as compared to controls (lack of consistent and dose dependent pattern of effect on food consumption in treated groups indicates no toxicological relevance).

2) Females
- no effect on food consumption was observed in treated groups during premating, gestation and lactation period as compared to controls.

HAEMATOLOGICAL FINDINGS:
- in male and female, no statistically significant effect was observed in any of the hematological parameters of treatment groups except statistically significant increase in red blood corpuscles and haematocrit in 250 mg/kg bw/day dose group when compared with controls.

- individual values for few haematological parameters in few animals were below or above the biological range as follows:
white blood corpuscules: in two females from control group, individual values were below the biological range (3.3-21.5 x 10³/μL)
red blood corpuscles: in one male from the 250 mg/kg bw/day dose group, individual value was slightly above the biological range (3.7-9.57 x 10^6/μL.
haematocrit: in one male from 250 mg/kg bw/day dose group, individual value was above the biological range (32-53.7 %).
mean corpuscular volume: in one female from 1000 mg/kg bw/day dose group, individual value was above the biological range (47.4-62 fL)
mean corpuscular haemoglobin concentration: in one male from 250 mg/kg bw/day dose group, individual value was below the biological range (30.8-37.7 g/dL).
- almost all individual and group mean values for total neutrophils in all treatment groups and control group were below the biological range (5-57 %).
- the majority of individual and mean partial prothrombin time values (29-51 sec) and activated partial prothrombin time values (15-32.2 sec) were below the biological range in males. In females only activated partial prothrombin time individual and mean values were below the biological range.

- absence of consistent and dose dependent pattern of effect on various haematological parameters indicates no toxicological relevance.

CLINICAL BIOCHEMISTRY FINDINGS:
- statistical analysis of clinical chemistry data revealed decrease in aspartat-aminotransferase in 1000 mg/kg bw/day dose group male and females (no toxicological significance and can not be attributed to the treatment).
- all other clinical chemistry parameters remained unaffected due the treatment with test item and were within the biological range.
- individual values and group mean values for glucose, cholesterol and total protein were below or above the biological range as follows:
glucose: in 3 control, 4 low and all 5 mid and high dose male animals, individual and mean values were above the biological range (1.7-15.3 mmol/L).
cholesterol: in 2 control, 3 low, 1 mid and 3 high dose group males, individual and mean value were below the biological range (1.02-14.6 mmol/L). In one female from each group, individual values were below the biological range. (1.02 - 14.6 mmol/L).
total protein: in 1 control, 2 mid and 4 high dose group males, individual values were below the biological range (52-80 g/L). In 1 control, 2 low, 2 mid and 1 high dose females, individual values were below the biological range (52-80 g/L).

BEHAVIOUR (FUNCTIONAL FINDINGS)
- no relevant differences were observed concerning functional examinations in male and females.
- 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.

ORGAN WEIGHTS
1) Males
- no statistically significant difference in the absolute and relative organ weights of the treatment groups were found except a decrease in relative heart weights in 250 mg/kg bw/day dose group when compared with the controls (considered to be incidental due to lack of dose dependency).
2) Females
- statistically significant increase in absolute and relative thymus weights was observed in 250 mg/kg bw/day dose group when compared with controls (considered to be incidental due to lack of dose dependency).

GROSS PATHOLOGICAL FINDINGS:
1) Males
- various macroscopic findings observed were left seminal vesicle diminished (one male; control), yellowish whitish deposition on epididymides (control group: one male; 250 mg/kg bw/day dose group: one male; 500 mg/kg bw/day: three males; 1000 mg/kg bw/day: one male), redish discolouration of lung (500 mg/kg bw/day: one male) and white spot on right seminal vesicle (1000 mg/kg bw/day: one male).
2) Females
- various macroscopic findings observed were slightly enlarged and discoloured left axillary lymph node (control: one female; 1000 mg/kg bw/day: one female), lungs with remains of the test item (250 mg/kg bw/day: three females; 500 mg/kg bw/day: two females; 1000 mg/kg bw/day: two females), small cyst on right kidney (500 mg/kg bw/day: one female), slightly reduced thymus (500 mg/kg bw/day: two females) and bloody, discoloured lung (500 mg/kg bw/day: one female; 1000 mg/kg bw/day: two females).
- all other males and females from various treatment groups did not show any macroscopic findings at necropsy.
- gross pathological observation of male and females at scheduled necropsy revealed no treatment related findings and therefore observed findings are considered to be spontaneous in nature.

HISTOPATHOLOGICAL FINDINGS - NON-NEOPLASTIC
- in the female and male reproductive organs, as well as in the other organs evaluated in this study, no histopathological lesions were found which were considered to be test item related.
- three females from the study (control: one female; 250 mg/kg bw/day: two females) were non-pregnant and showed histopathologically metestrus/diestrus cycle in the sexual organs.
- in the lung, blackish intra-alveolar material, together with foci of pneumonitis or diffuse pneumonia, was noted in a low number of animals, mostly females, and was considered to represent misgavaged or aspirated test item.

REPRODUCTIVE FUNCTIONS - SPERM MESURES
- no treatment-related findings were made for testis and epidymidis weight
- no treatment-related findings were made during histopathology

REPRODUCTIVE PERFORMANCE:
- 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, 9, 8 and 10 pregnancies in control, 250, 500, and 1000 mg/kg bw/day dose groups, respectively.
- reduced fertility index was observed in control (90%) and 500 mg/kg bw/day dose group (80 %) as compared to 250 mg/kg bw/day (100 %) and 1000 mg/kg bw/day dose groups (100 %).
- group mean number of corpora lutea, number of implantation sites, percent preimplantation loss and post implantation loss remained unaffected due to treatment when compared with controls. Although there was a slight increase in preimplantation loss in treated groups as compared to controls, there was no statistical significance therefore this effect can not be attributed to the treatment.
-no treatment related effect on copulation index, fertility index, and delivery index was observed when compared with controls.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

- one male pup from a female as well as one male and one female pup from another female of the 1000 mg/kg bw/day dose group were found dead on PND 1. Gross necropsy findings of found dead pup revealed no specific findings.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

MORTALITY / VIABILIY:
- number of live pups born on post natal day 0,remained unaffected due to treatment when compared with controls.
- no treatment related effect on viability index was observed when compared with controls.
- no statistically significant effect on survival of the pups from post natal day (PND) 0 to PND 4 was observed in any treatment group when compared with controls.

BODY WEIGHT AND WEIGHT CHANGES:
- statistical analysis of litter weight data revealed decrease in male litter weight on post natal day (PND) 0 in 250 and 500 mg/kg bw/day dose groups and on PND 4 in l250 mg/kg bw/day dose group. 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.

GROSS PATHOLOGICAL FINDINGS:
- no macroscopic observations were observed at necropsy.

OTHER EFFECTS:
- statistically significant decrease in No. of male pups and sex ratio was observed in 250 mg/kg bw/day dose group on post natal day (PND) 0 and 4.
- number of still births and runts were observed in 1000 mg/kg bw/day dose group. Although this effect was not statistically significant, due to the dose dependency, it could be attributed to the treatment.
- 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.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:

not specified

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

After oral administration of 250, 500, and 1000 mg/kg bw/day of diniobium pentaoxide, no adverse test item related effects were observed for clinical signs, mortality, body weight, body weight changes, food consumption, haematology, clinical chemistry, neurobehavioural examinations, organ weights, gross pathology, and histopathology. Lastly, no adverse test item related effects could be determined for reproductive function (sperm measures) and reproductive performance in male and female rats.

Under the experimental conditions of this screening study, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity and reproduction/developmental toxicity was considered to be ≥ 1000 mg/kg bw/day for male and female rats.