Diniobium pentaoxide

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 Dec 2021 - 14 June 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Remarks:

14-day inhalation dose rangefinder study

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

13 Oct - 28 Oct 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to other study

Remarks:

90-day inhalation repeated dose toxicity study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study

Version / remarks:

adopted 25 June 2018

Deviations:

yes

Remarks:

see field "Principles of method if other than guideline"

Principles of method if other than guideline:

The study was a 14-day dose rangefinder for the main 90-day inhalation study, therefore, only assessed a limited number of parameters: clinical observations, body weights, gross pathology, histopathology (left lung lobe, including bronchi and the lung-associated lymph nodes (LALN, mediastinal and tracheobronchial), trachea, larynx, pharynx and the nasal cavities (including NALT) in all animals of the control and high dose group) and bronchoalveolar lavage (BAL).

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI (Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland (Sulzfeld, Germany)
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: males approx. 250 g and females approx. 175 g
- Housing: Animals were housed in Makrolon® (polycarbonate) cages type IV, five rats per cage. Cages and absorbing softwood ('ssniff BK 8-15') bedding material was changed once a week or more often, if necessary.
- Diet: Commercial chow in pellet form (Ssniff V1534, Ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum.
- Water: Tap water from the Hannover city water supplier, ad libitum.
- Acclimation period: For a period of 2 - 3 weeks prior to exposure animals were trained to become accustomed to nose-only tubes.

DETAILS OF FOOD AND WATER QUALITY: A certificate of water analysis issued by the water supplier (Stadtwerke Hannover) was sent periodically to test facility. A certificate of feed analysis was issued by the supplier periodically. There were no known contaminants in the feed or water at levels that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): not provided
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

1.4

Geometric standard deviation (GSD):

1.8

Details on inhalation exposure:

Aerosol Generation:
The particulate sample aerosols were generated by dispersing the dry powder. Dispersion was achieved by a feeding system and a high-pressure, high-velocity pressurized air dispersion nozzle developed by the test facility.
For each nose-only exposure unit, the aerosol was generated by a high-pressure pneumatic disperser. The disperser was fed with the test substance under computerized control, i.e. with a feed-back loop to the actual aerosol concentrations measured by an aerosol photometer (see Figure 1 attached).
The photometer gives a scattering light signal which is proportional to the particle concentration, if the particle size distribution is constant. The ratio between photometer signal and concentration was determined throughout the study by comparing to gravimetric concentrations.
The aerosol was given to the rats by a flow-past nose-only inhalation exposure system. In this system, aerosols were supplied to each rat individually, and exhaled air was immediately exhausted. The airflow to each rat was approximately 1 L/min which was calculated to be laminar. Therefore measurement of the oxygen concentration was not necessary. Prior to the 14-day exposure of rats, technical trials to adjust particle size distributions and exposure levels were conducted.
Additionally, the MMAD was determined twice (once before exposure start and once during the exposure period) for each test item exposure unit (3 units) by a cascade impactor (Marple impactor). Filter samples of the aerosols were taken once per day to control the aerosol concentrations and to calibrate the aerosol photometers. These samples were collected at a port of the nose-only exposure unit, thus, under the same conditions the rats are inhaling the aerosol.
Air flow, temperature and relative humidity were measured continuously and recorded by 20-minute means

Exposure of Rats
For exposure to the test item the rats were restrained in acrylic tubes with a flexible stopper. The exposure tubes were arranged around a cylinder capable to take up 16 tubes per platform. The rat nose was located at the front end of a tube being connected to a cylinder delivering the aerosol. Through the thin pipes, the aerosol was supplied to each rat nose individually and exhaled air was drawn off immediately by a cylinder surrounding the aerosol delivering cylinder. The position of exposure tubes of rats at the cylinder was changed daily according to a rotation plan to minimize exposure differences due to geometry. The exposure units (4 units) were located each under a separate hood to prevent contamination among different dose groups.
The duration of exposure was 6 hours/day, 5 days/week for 2 weeks.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The aerosol concentrations were measured by an aerosol photometer.
The photometer gives a scattering light signal which is proportional to the particle concentration, if the particle size distribution is constant. The ratio between photometer signal and concentration was determined throughout the study by comparing to gravimetric concentrations.
The aerosol concentrations and MMAD values are provided in Tables 1 and 2 (see field "Any other information on materials and method incl. tables").

Duration of treatment / exposure:

14 days

Frequency of treatment:

6 h/day, 5 days/week

Dose / conc.:

3.82 mg/m³ air (analytical)

Remarks:

target aerosol concentration = 3.5 mg/m3

Dose / conc.:

13.96 mg/m³ air (analytical)

Remarks:

target aerosol concentration = 14 mg/m3

Dose / conc.:

58.96 mg/m³ air (analytical)

Remarks:

target aerosol concentration = 56 mg/m3

No. of animals per sex per dose:

5

Control animals:

yes

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- All animals were clinically observed in their cages at least once a day. During the exposure, the animals were clinically observed continuously at the beginning and then periodically at intervals of 15 to 30 minutes. In addition, observation took place both before and after exposure at regular intervals.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week
- Cage side observations checked: inspection of skin, fur, eyes, visible mucous membranes, examination for pathomorphological changes (e.g. unusual breathing pattern, masses, nodules), abnormal behaviour and central nervous symptoms (e.g. changes in gait, posture or grooming activity, unusual response to handling, secretion/excretion abnormalities, clonic/tonic movements, stereotypies) and/or other clinical abnormalities.

BODY WEIGHT: Yes
- Time schedule for examinations: twice weekly

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: after the end of the 14 day exposure period (post exposure Day 1)
- Dose groups that were examined: all groups
- Number of animals: 5 per sex
- Parameters checked:
Cytological parameters
• total cell count (recruitment of lung leukocytes)
• differential cell count (inflammatory (PMNs) or immunological (lymphocytes) reactions)
Biochemical parameters
• lactic dehydrogenase (LDH = cytosolic marker enzyme; increased permeability of membranes, cell damage and lysis)
• β-glucuronidase (measure of phagocytic activity of macrophages; lysis of macrophages)
• total protein (marker of transsudation; damage of epithelial cells)

LUNG BURDEN: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals were subjected to a complete necropsy, which included careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents.
All rats were anesthetized with an overdose pentobarbital sodium (Narcoren®) and killed by cutting the vena cava caudalis.
The abdominal cavity was opened and the diaphragm was cut carefully allowing the lungs to collapse. Heart, esophagus, upper half of trachea, thymus and lung associated lymph nodes (LALN; mediastinal and tracheobronchial) were removed from the lung convolution.
The lung and the lower half of the trachea were weighed. The left lung was used for histopathology and the right lung for BAL. For histopathology, the lung was inflated under a pressure of about 20 cm water with formalin and was fixed by immersion for a minimum of 2 hours, and used for histopathology.
The respiratory tract was preserved as follows: Nasal passages (including nasal -associated lymphoid tissue-NALT), larynx, trachea, lungs, and LALN (mediastinal and tracheobronchial). All tissues listed in OECD Guideline no. 413 were preserved for histopathology.

HISTOPATHOLOGY: Yes
The following histopathology was performed in 5 animals per group and sex after the end of exposure:
• Histopathology of left lung lobe, including bronchi and the lung-associated lymph nodes (LALN, mediastinal and tracheobronchial), trachea, larynx, pharynx and the nasal cavities (including NALT) in all animals of the control and high dose group.
• Trimming of lungs: left lung lobe; nose: 4 sections.
• Histopathology of other organs only if specifically indicated (e.g. by macroscopical findings).
Lungs were fixed in buffered formalin (10%), embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H & E).

Statistics:

Differences between groups were considered statistically significant at p < 0.05. Data were analyzed using analysis of variance. If the group means differed significantly by the analysis of variance the means of the treated groups were compared with the means of the control groups using Dunnett's test (Dunnett, 1955; 1964).
The statistical evaluation of the histopathological findings were done with the two-tailed Fisher test by the PROVANTIS system. If necessary, further statistical procedures were applied upon agreement with the sponsor.

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical observations outside the normal range were recorded.

Mortality:

no mortality observed

Description (incidence):

All animals survived the study and tolerated well the exposure to the test item at all concentrations.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Statistically significant changes were not observed in the treatment groups as compared to controls.

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

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Means of terminal body, absolute and relative lung weights did not show any statistically significant changes in the diniobium pentaoxide low, mid and high dose group as compared to clean air controls.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Findings related to the inhalation of the test item were detected within the lung, the nasal cavity and the lung-associated lymph-nodes.

Clean air control group (group 1)
In the laryngopharynx, the lung-associated lymph nodes, the nasopharynx and the trachea, no pathologic findings were observed.
In the lung, in 2/5 males and 1/5 females no visible lesions were found. In 3/5 males and 4/5 females, a multifocal very slight perivascular granulocytic cell infiltration was observed.
In the larynx, there was a slight focal granulomatous inflammation accompanied by a focal slight plant inspissation in 1/5 male rats. In addition, 1/5 male rats exhibited a very slight focal subepithelial mononuclear cell infiltration.
In the nasal-cavity, a very slight multifocal subepithelial mixed inflammatory cell infiltration was seen in 1/5 male rats.
All these lesions represent commonly found background lesion in the lungs of these rat strain.

Diniobium pentaoxide high dose group
In the laryngopharynx, the nasopharynx and the trachea, no pathologic findings were observed.
In the lung, exposure-related findings represent a multifocal accumulation of particle-laden macrophages in the alveoli in all male (1 slight, 4 moderate) and all female (1 slight, 4 moderate) rats. Further, there was a very slight multifocal accumulation of particle-laden macrophages in the bronchus-associated lymphoid tissue (BALT) in 4/5 males and 5/5 females. Particles were also observed free (extracellular) in 5/5 male (1 very slight, 4 slight) and in 5/5 female (1 very slight, 4 slight) rats.
Interpreted to be unrelated to the inhalation of the test item, there was a multifocal very slight perivascular granulocytic cell infiltration in 3/5 males and 4/5 females.
In the nasal cavity, exposure-related findings represented a very slight multifocal accumulation of particle-laden macrophages in the nose-associated lymphoid tissue (NALT) in 5/5 males and 5/5 females.
Interpreted to be unrelated to the exposure, a multifocal very slight mucous cell hyperplasia was observed in 1/5 males.
In the lung associated lymph nodes (LALN), exposure-related findings represented a very slight multifocal accumulation of particle-laden macrophages in 2/5 males and 2/5 females.
In the larynx, interpreted to be unrelated to the inhalation of the test item, 1/5 male rats exhibited a very slight focal subepithelial mononuclear cell infiltration.

In summary, exposure-related findings were detected within the lung, the nasal cavity and the lung-associated lymph-nodes. These findings consisted of accumulation of particle-laden macrophages within the alveoli and the bronchus-associated lymphoid tissue in the lung, within the nose-associated lymphoid tissue in the nasal cavity and within the lung-associated lymph nodes. Further, free (extracellular) particles were also observed in the lung. These findings were interpreted as adaptive non-adverse findings. Additional exposure-related findings were not seen.
In conclusion, within the investigated diniobium pentaoxide high dose group, no adverse histopathological findings were observed after a 14-day nose-only inhalation toxicity study (dose range finding study).

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Cytological parameters:
At Day 1 post-treatment no statistically significant and treatment-related alterations in the percentages of polymorphonuclear neutrophils (PMN) were observed in the Nb2O5 low, mid and high dose treated groups in a dose dependent manner. The test item concentrations did not induce an adverse PMN-related lung inflammation. Overall, the collected data show values at the level of the control.
Biochemical parameters:
For lactate dehydrogenase, ß-glucuronidase and total protein no statistically significant increases were detected in the control and the Diniobium pentaoxide treated groups. These results mirror the PMN data, which showed no statistically significant increase in any treatment group.

Dose descriptor:

NOAEC

Effect level:

59 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Conclusions:

In this 14-day nose-only inhalation dose rangefinder study, no clear differences of the inflammogenic potential were observed among the three different dosages of diniobium pentaoxide. The NOAEC was estimated to be ≥ 59 mg/m3 based on the polymorphonuclear neutrophil values analysed in the bronchoalveolar lavage fluid analysis and the absence of histopathological findings.

Based on this study, dose levels of 1.5, 6 and 24 mg/m3 were selected for the 90-day nose-only inhalation study. For the high dose group, a lung overload will be achieved following 90 days of exposure (with respect to retained mass or volume in lungs).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study

Version / remarks:

adopted 25 June 2018

Deviations:

yes

Remarks:

ophthalmology was not included as this endpoint is not sensitive in particle studies.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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(5+) pentaoxidandiide

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI (Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland (Sulzfeld, Germany)
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 9 to 10 weeks
- Weight at study initiation: males approx. 250 g and females approx. 200 g
- Fasting period before study: no
- Housing: Animals were housed in Makrolon® (polycarbonate) cages type III, and were maintained under conventional laboratory conditions. Cages and absorbing softwood ('ssniff BK 8-15') bedding material were changed once a week or more often, if necessary.
- Diet: Commercial chow in pellet form (Ssniff V1534, Ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum.
- Water: Tap water from the Hannover city water supplier, ad libitum.
- Acclimation period: Acclimatisation was approximately one week when the animals were allowed to adjust to the Fraunhofer ITEM environment. During the 2 - 3 weeks prior to exposure start, all rats were trained to the 6-hour restraint in nose-only tubes.
Clinical observations were made every day. Body weight was measured during the acclimatization period.

DETAILS OF FOOD AND WATER QUALITY: A certificate of water analysis issued by the water supplier (Stadtwerke Hannover) was sent periodically to test facility. A certificate of feed analysis was issued by the supplier periodically.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): not stated
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

1.4

Geometric standard deviation (GSD):

1.8

Details on inhalation exposure:

Aerosol Generation:
The test item was aerosolized using a dry dispersion system optimized for powdered substances and operated with pressurized air (Figure 1, attached background material). Cyclones (in line) were used to reduce the coarse moiety of the aerosol. The signal of an aerosol photometer was used to control the feed rate of the dispersion system in order to keep the aerosol concentration in the inhalation unit constant. Actual test item concentrations were measured in the breathing zone of the animals.
For each nose-only exposure unit, the aerosol was generated by a high-pressure pneumatic disperser. The disperser was fed with the test substance under computerized control, i.e. with a feedback loop to the actual aerosol concentrations measured by an aerosol photometer (see Figure 1, attached background material).
The photometer gives a scattering light signal which is proportional to the particle concentration, if the particle size distribution is constant. The ratio between photometer signal and concentration was determined throughout the study by comparing to gravimetric concentrations.
The aerosol was given to the rats by a flow-past nose-only inhalation exposure system. In this system, aerosols were supplied to each rat individually, and exhaled air were immediately exhausted. The airflow to each rat was approximately 1 L/min which is calculated to be laminar. Therefore, measurement of the oxygen concentration was not necessary. Prior to the 90-day exposure of rats, technical trials to adjust particle size distributions and exposure levels were conducted.

Monitoring and Controlling the Exposure Atmospheres:
Air flow, temperature and relative humidity were measured continuously and recorded by 20-minute means.
Additionally, the MMAD was determined four times (once before exposure start and once per month during the exposure period for each test item exposure unit (3 units) by a cascade impactor (Marple impactor). Filter samples of the aerosols were taken once per day to control the aerosol concentrations and to calibrate the aerosol photometers. These samples were collected at a port of the nose-only exposure unit, thus, under the same conditions the rats were inhaling the aerosol. The evaluation of filter samples were done by gravimetrical analysis. As a permanent control of the aerosol concentrations is guaranteed by photometers the scheduled filter sampling frequency was sufficient (in agreement with OECD guideline 413).

Exposure of Rats:
For exposure to the test item the rats were restrained in acrylic tubes with a flexible stopper. The exposure tubes were arranged around a cylinder capable to take up 16 tubes per platform. The rat nose was located at the front end of a tube being connected to a cylinder delivering the aerosol. Through the thin pipes, the aerosol was supplied to each rat nose individually and exhaled air was drawn off immediately by a cylinder surrounding the aerosol delivering cylinder. The position of exposure tubes of rats at the cylinder was changed daily according to a rotation plan to minimize exposure differences due to geometry. The exposure units (4 units) were located each under a separate hood to prevent contamination among different dose groups.
The duration of exposure was 6 hours/day, 5 days/week for 13 weeks; subsequently, a clean air recovery period up to 90 days followed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Details of the analytical methods are described in the field "Details on inhalation exposure".

For the analytical results, please refer to the tables in the field "Any other information on material and methods incl. tables":
Mean aerosol concentrations determined by gravimetry (Table 1)
Gravimetrically recorded daily aerosol concentrations (Table 2)
Results of MMAD determination (Table 3)

Duration of treatment / exposure:

90 days (+ up to 90 days post-exposure observation)

Frequency of treatment:

6 h/day, 5 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1.5 mg/m³ air (analytical)

Remarks:

target aerosol concentration = 1.5 mg/m3

Doses / concentrations 2

Dose / conc.:

5.99 mg/m³ air (analytical)

Remarks:

target aerosol concentration = 6 mg/m3

Doses / concentrations 3

Dose / conc.:

24.01 mg/m³ air (analytical)

Remarks:

target aerosol concentration = 24 mg/m3

No. of animals per sex per dose:

25 males and 15 females

For an overview of the exposure groups, see Table 4 (Any other information on materials and methods incl. tables).

Control animals:

yes

Details on study design:

- Dose selection rationale:
In the 14-day nose-only inhalation dose rangefinder study (supporting study, Bruer, G.), no clear differences of the inflammogenic potential were observed among the three different dosages of diniobium pentaoxide. The NOAEC was estimated to be ≥ 59 mg/m3 based on the polymorphonuclear neutrophil values analysed in the bronchoalveolar lavage fluid analysis and the absence of histopathological findings.
Based on this study, dose levels of 1.5, 6 and 24 mg/m3 were selected for the 90-day nose-only inhalation study. For the high dose group, a lung overload will be achieved following 90 days of exposure (with respect to retained mass or volume in lungs).
- Fasting period before blood sampling for clinical biochemistry: not specified
- Rationale for selecting satellite groups: satellite groups were included to assess recovery of any test item related findings and to assess clearance / retention / disposition of the test item in the lungs.
- Post-exposure recovery period in satellite groups:
28 days for the assessment of lung burden in males.
90 days for the assessment of lung burden and histopathology in males and histopathology and bronchoalveolar lavage in females.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were clinically observed in their cages at least once a day (with the exception of weekends and public holidays: once daily). During exposure, animals were regularly observed at periodic intervals. In addition, observation took place after exposure until end of work.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before sacrifice, animals were inspected outside their home cages and carefully examined for clinical symptoms, i.e. abnormalities concerning their general condition. This included inspection of skin, fur, eyes, visible mucous membranes, examination for pathomorphological changes (e.g. unusual breathing pattern, masses, nodules), abnormal behaviour and central nervous symptoms (e.g. changes in gait, posture or grooming activity, unusual response to handling, secretion/excretion abnormalities, clonic/tonic movements, stereotypies) and/or other clinical abnormalities.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weight was recorded to the nearest 0.1 g on day -7 before treatment and twice a week in the first 4 weeks and once a week thereafter throughout the study for all animals.

FOOD CONSUMPTION:
- Food consumption was recorded weekly during the study period (including post-exposure observation period) using 2 to 10 animals per sex and dose group, depending on the date.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was recorded weekly during the study period (including post-exposure observation period) using 2 to 10 animals per sex and dose group, depending on the date.

OPHTHALMOSCOPIC EXAMINATION: No (this endpoint is not sensitive in particle studies)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 1 after the 90-day exposure period.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: 10 animals per sex per dose group.
- Parameters examined: Red blood cells (RBC); Total white blood cells (WBC); Haemoglobin (HB); Differential white cell count (% and absolute*); Haematocrit (HCT); Platelets (PTL); Reticulocytes (RET); Mean cell volume (MCV)*; Mean haemoglobin/erythrocyte (MCH)*; Mean hemoglobin concentration/erythrocyte (MCHC)*; Prothrombin time (PT) and thromboplastin time (TP)
* = calculated values

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 1 after the 90-day exposure period.
- Animals fasted: Not specified
- How many animals: 10 animals per sex per dose group.
- Parameters examined: Aspartate aminotransferase (AST); Alanine aminotransferase (ALT); Alkaline phosphatase (AP); γ-Glutamyl transpeptidase (GGT); Urea; Triglycerides; Total bilirubin; Creatinine (CREA); Total protein (TP); Albumin (ALB); Globulin (GLB)*; ALB/GLB (A/G)*; Glucose (GLUC); Cholesterol (CHOL); Sodium (Na); Calcium (Ca); Potassium (K); Phosphorus (PO4); Chloride (CL);
* = calculated values

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: Bronchoalveolar lavage was performed in 10 rats per sex and group 1 day after last exposure and following a 90-day period of recovery in 5 female rats per group.
- Dose groups that were examined: all dose groups.
- Parameters examined:
Cytological parameters: total cell count (recruitment of lung leukocytes); differential cell count (inflammatory (polymorphonuclear neutrophils) or immunological (lymphocytes) reactions). A total of 400 leukocytes per rat were evaluated.
Biochemical parameters: lactic dehydrogenase (LDH = cytosolic marker enzyme; increased permeability of membranes, cell damage and lysis); β-glucuronidase (measure of phagocytic activity of macrophages; lysis of macrophages); total protein (marker of transudation; damage of epithelial cells).

LUNG BURDEN: No
Due to the unremarkable findings of the BALF analysis and the histopathological examinations, no lung burden analysis was performed and the lungs were preserved for further analysis if necessary.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals were subjected to a complete necropsy, which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
The rats used for histopathological and BAL investigations were anesthetized with an overdose pentobarbital sodium (160 mg / mL, 1 mL / kg body weight i.p., Narcoren®) and exsanguinated by cutting the vena cava caudalis. For the determination of prothrombin time at final sacrifice, 900 µL blood was drawn from the vena cava using a syringe with 100 µl 10% (v/v) of citric acid sodium salt (0.11 mmol/l).
The abdominal cavity was opened and the diaphragm was cut carefully allowing the lungs to collapse. Heart, esophagus, upper half of trachea, thymus and lung associated lymph nodes (LALN) were removed from the lung convolution.
The lung and the lower half of the trachea were weighed (= lung wet weight) and used for BAL (right lobes no. 1–4*) or histopathology (left lobe no. 5*).
For histopathology the left lung lobe no. 5* was inflated under a pressure of about 20 cm water with formalin and was fixed by immersion for a minimum of 2 hours and used for histopathology.
Histopathology was only performed for the control and diniobium pentaoxide high group. However, organs of all groups were fixed in formalin for further analysis if required.
The following organs were preserved and wet weights were recorded: liver, kidneys, adrenals, testes, epididymides, ovaries, uterus, thymus, spleen, brain, lung and heart. The respiratory tract was preserved as follows: Nasal passages (including nasal-associated lymphoid tissue (NALT)), larynx, trachea, lungs, and LALN (mediastinal and tracheobronchial). All tissues listed in OECD 413 were preserved for histopathology (Table 5) excluding those in brackets and the seminal vesicles were prepared for histopathology. Tissues in brackets were preserved only.

HISTOPATHOLOGY: Yes
The following histopathology was performed in 10 animals per sex of the control and diniobium pentaoxide high dose group (Groups 1 and 4) at Day 1 post-exposure:
- Histopathology of the left lung lobe at three levels, including main bronchi, and the LALN from the hilar region of the lung (mediastinal and tracheobronchial), trachea, larynx, pharynx and the nasal cavities (including NALT).
- Trimming: nose 5 sections (see Figure 2*; the section 5 included the olfactory bulb in situ); left lung lobe (see Figure 3*).
- Histopathology of the other organs according to Table 5.
The lung lobe was fixed in buffered formalin (10%) for up to one day, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE). In addition, masson trichrome staining was used for detection of connective tissue production within the lung.
For histological examination of the other organs of the respiratory tract, tissues were fixed for at least one day in buffered formalin (10%), embedded in paraffin, sectioned, and stained with HE. Bones were decalcified prior to embedding.

* See Figures, included as attached background material.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical observations did not show any signs of systemic toxicity in the animals.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed in the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No relevant statistically significant changes were observed in the treatment groups as compared to concurrent controls (see Attached background material: Figures 3 and 4. Mean data are presented in Appendix 14.2. Individual data are shown in Appendix 14.3).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

In both sexes, no relevant statistically significant changes as compared to clean air controls were observed.
Means of food consumption data in g/day are illustrated in Figures 6 and 7 and presented in Appendix 14.4 (Attached background material).

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

In both sexes, no relevant statistically significant changes as compared to clean air controls were observed.
Means of water consumption data in g/day are illustrated in Figures 8 and 9 and presented in Appendix 14.5 (Attached background material).

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

In both sexes, no relevant statistically significant dose-dependent alterations were observed.
Mean values are shown in Table 12 and individual data in Appendix 14.7 (Attached background material).

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

In both sexes, no relevant statistically significant dose-dependent alterations were observed.
Mean values are shown in Table 13 and individual data in Appendix 14.8 (Attached background material).

Endocrine findings:

not specified

Description (incidence and severity):

The following ED-related parameters were investigated in the study:
Coagulating gland histopathology, Epididymis histopathology, Epididymis weight, Liver weight, Mammary gland histopathology (males and females), Ovary histopathology, Ovary weight, Oviduct histopathology, Prostate histopathology, Seminal vesicles histopathology, Testis histopathology, Testis weight, Thyroid histopathology, Thyroid weight, Uterus histopathology (with cervix), Uterus weight, Vagina histopathology, Adrenals histopathology, Adrenals weight, Brain weight, Pituitary histopathology
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):

Absolute and relative lung wet weights did not result in relevant statistically significant changes in any treatment group. Mean data are presented in Table 10 and 11 (attached background material), individual data are shown in Appendix 14.6 (Attached background material).
Statistical significance for other organs occurred sporadically and are therefore considered negligible.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

All animals were sacrificed at scheduled dates. Upon necropsy, enlarged lung-associated lymph nodes (LALN) were observed in some animals distributed over some treatment groups. This could be a particle-specific lung clearance pathway. It was not considered to be the case in this instance due to the small number of animals and the distribution across different groups.
Lungs did not show any typical dose-dependent findings caused by the test item.
No other test item- or dose-related gross findings were detected. Only some incidental macroscopic observations were obtained.
See Table 9 (Attached background material) for a summary of gross findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Clean air control group:
In the lung, interpreted to be unrelated to the exposure, there was a perivascular infiltration of granulocytes in 8/10 males (7 very slight; 1 slight) and 7/10 females (all very slight), which represented a commonly found background lesion in this rat strain, which partially correlated with the macroscopically observed small white areas.
Single lesions were seen in different organs, which represented commonly found background lesions in this rat strain.

Diniobium pentaoxide high dose group:
The only exposure-related finding were an accumulation of particle-laden macrophages as well as intracellular and free particle in different parts of the respiratory tract.
In the lung, exposure-related findings presented as particle-laden macrophages, multifocal in the alveoli in 10/10 males (9 moderate; 1 severe) and 10/10 females (2 slight; 5 moderate; 3 severe) as well as in the bronchus-associated lymphoid tissue in 10/10 males (7 very slight; 3 slight) and in 10/10 females (9 very slight; 1 slight). Further, particles were seen multifocally in small amounts (very slight) intracellular in the alveoli and interstitium (in one male slight) as well as free within the alveoli in all male and female (10/10 each) animals. There was an increase of pneumocytes type 2 (pneumocyte type 2 hyperplasia) multifocal very slight in all male and female rats (10/10 each).
The multifocal very slight perivascular infiltration of granulocytes in 5/10 males and 3/10 females represent a commonly found background lesion in this rat strain and were interpreted to be unrelated to the treatment.
In the nasal cavity, a very slight multifocal accumulation of particle-laden macrophages was found in 9/10 males and 10/10 females in the nose-associated lymphoid tissue (NALT).
In the lung-associated lymph nodes (LALN), there was a multifocal accumulation of particle-laden macrophages in 9/10 males (4 very slight; 3 slight; 2 moderate) and in 9/10 (2 very slight; 5 slight; 2 moderate) females.
In the trachea, there was a (multi)focal very slight accumulation of particle-laden macrophages in 1/10 males and in 2/10 females.
All the other findings within the different organs occurred in single animals and were interpreted to be incidental without any relation to the exposure.
See Appendix 14.12 for the histopathology report (Attached background material).

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

One control animal showed a benign skin tumour (papilloma, squamous cell) at the upper lip. This tumour was interpreted to be incidental.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Bronchoalveolar lavage (BAL)
Cytological and biochemical parameters:
Mean values are shown in Figures 10–12 and Table 14 and individual data in Appendix 14.9 (Attached background material).
At Days 1 and 90 post-exposure, no relevant statistically significant increases of polymorphonuclear neutrophils (PMNs) were detected in male treatment groups on Day 1 and in female treatment groups on Day 90. The PMN percentages (in the range from 2.7% to 6.6%, males – 0.1% to 2.4%, females) were close to historical clean air control data; thus, the test item did not induce a relevant PMN-related lung inflammation. On Day 1 a slight significant increase of PMN was detected in the female high dose group (13.8 %). This increase indicated a PMN-related lung inflammation, but did not appear to be relevant in the context of the lack of histopathological findings. Lymphocyte levels were also found to be low and at control levels (<1%).
For lactic dehydrogenase, ß-glucuronidase and total protein, no relevant statistically significant increases were detected in any group of both sexes and sacrifice dates (Table 15, Attached background material).

Lung weights of rats used for BAL:
Relative lung weights of rats used for BAL were statistically significantly increased in the female mid and high dose treatment group on Day 1 post exposure. Absolute lung weights were not increased in any treatment group in comparison to historical control data (males absolute: approx. 1.3 g – females approx. 1.1 g). Based on this finding and the absence histopathological findings, the increased relative lung weights in female groups appeared to be negligible.
The mean data are compiled in Table 16 and the individual data is shown in Appendix 14.10 (Attached background material).

Lung weights of rats used for burden analysis:
Due to the unremarkable findings of the BAL analysis and the histopathological examinations, no lung burden analysis was performed and the lungs were weighed and preserved.
Absolute and relative lung weights of male rats used for lung burden analysis were not statistically significantly increased in any treatment group.
The mean data are compiled in Table 17 and the individual data is shown in Appendix 14.11 (Attached background material).

Effect levels

Key result

Dose descriptor:

NOAEC

Effect level:

>= 24 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at this dose level (highest dose level tested)

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

This sub-chronic inhalation toxicity study was conducted in accordance to OECD 413 and in compliance with GLP. Exposure-related findings were detected in the investigated diniobium pentaoxide high dose group within the lung, the nasal cavity, the lung-associated lymph nodes and the trachea.
The accumulation of particle-laden macrophages in the alveoli and bronchus-associated lymphoid tissue, the intracellular and free particles as well as the pneumocyte type 2 hyperplasia were interpreted to be non-adverse adaptive changes in the lung. Similarly, the accumulation of particle-laden macrophages in the lung-associated lymph nodes, in the trachea and in the nose-associated lymphoid tissue of the nasal cavity were also interpreted to be non-adverse adaptive changes.
No additional exposure-related changes were seen in the respiratory tract.
In conclusion, no adverse findings were detected within the investigated diniobium pentaoxide high dose group and the NOAEC was determined to be ≥ 24 mg/m3.