Diniobium pentaoxide

Endpoint summary

Description of key information

Diniobium pentaoxide is not bioavailable:

Repeated dose oral toxicity (OECD 422): NOAEL ≥ 1000 mg/kg bw/day.

Repeated dose inhalation toxicity (OECD 413): NOAEC ≥ 24 mg/m³.

 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 September 2009 to 12 April 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

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

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

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

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

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Dosage
Based on the available information from other toxicity studies and in consultation with sponsor following doses were selected:
Control: 0 mg/kg bw/day
LD: 250 mg/kg bw/day
MD: 500 mg/kg bw/day
HD: 1000 mg/kg bw
The highest dose level was chosen with the aim of inducing toxic effects but not death or severe suffering. Thereafter, a descending sequence of dose levels is selected with a view to demonstrate any dosage related response and no observed adverse effect (NOAEL).
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle in the volume same as treated groups.

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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

Duration of treatment / exposure:

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

Frequency of treatment:

7 days per week

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes

Positive control:

no

Observations and examinations performed and frequency:

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

Sacrifice and pathology:

Males were sacrificed after the completion of mating period (total dosing of 28-29 days) and females were sacrificed on respective post natal day 4 along with pups by using high dose of sodium pentobarbital.
Dead pups and pups sacrificed at day 4 post-partum, or shortly thereafter were carefully examined for gross abnormalities.

Other examinations:

Litter observations
The duration of gestation was recorded and was calculated from day 0 of pregnancy. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.
Live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Live Pups were identified by writing actual numbers on the back with the help of permanent marker In addition to the observations on parent animals; any abnormal behaviour of the offspring was recorded.

Haematology
The following haematological examinations were made in five males and five females randomly selected from each group.
Haematocrit, haemoglobin, erythrocyte count, total and differential leucocyte count,
platelet count, blood clotting time.
Blood samples were taken from abdominal aorta as a part of the procedure for killing the animals, and stored under appropriate conditions.

Clinical Biochemistry
Clinical biochemistry determinations to investigate major toxic effects in tissues and, specifically, effects on kidney and liver were performed on blood samples obtained from the randomly selected five males and five females of each group. Investigations of plasma or serum included sodium, potassium, glucose, total cholesterol, urea, creatinine, total protein and albumin, two enzymes indicative of hepatocellular effects (such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase).
Urinalysis was not performed on the samples collected from five randomly selected males at the terminal sacrifice.

Gross Pathology
Males were sacrificed after the completion of mating period (total dosing of 28-29 days) and females were sacrificed on respective post natal day 4 along with pups by using high dose of sodium pentobarbital. However, in the animals randomly selected for the blood collection, anaesthesia solution (Ketamin/Xylazin, 2:1) was used. At the time of sacrifice or death during the study, the adult animals were subjected to a full, detailed gross necropsy which included careful 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.
The number of implantation sites and corpora lutea was recorded for each lactating female at necropsy.
Dead pups and pups sacrificed at day 4 post-partum, or shortly thereafter were carefully examined for gross abnormalities.
The 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. Tissues were fixed in 10% formalin with the exception of testes and epididymides which were fixed in modified Davidson's solution.

Organ Weight
Reproductive organs from all animals were weighed (testes, epididymides, prostate, seminal vesicle with coagulating glands as whole, ovaries, uterus with cervix as applicable). Apart from the reproductive organs from all animals, 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 in 10% formalin as it is the most appropriate medium for both the type of tissue and the intended subsequent histopathological examination.
- all gross lesions
- brain (representative regions including cerebrum, cerebellum and pons)
- spinal cord
- liver
- kidneys
- adrenals
- stomach
- small and large intestines (including Peyer´s patches)
- thymus
- thyroid
- spleen
- trachea and lungs (preserved by inflation with fixative and then immersion)
- heart
- urinary bladder
- lymphnodes (one lymph node covering the route of administration (mandibular) and another one distant from the route of administration to cover systemic effects (mesenteric))
- peripheral nerve (e.g. N. ischiadicus/ N. tibialis) in close proximity to muscle
- section of bone marrow

Histopathology
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. On the HE (Haematoxylin and Eosin) stained slides of all preserved organs and tissues. These examinations were not extended to animals of all other dosage groups as no treatment related changes were observed in the high dose group.
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 at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.
Histopathological evaluation and processing was performed at GLP-certified test site KALEIDIS –Consultancy in Histopathology, 6 rue du Gers, 68300 Saint-Louis, France. Blocking, embedding, cutting, staining and professional evaluation was performed according to the corresponding SOPs of the test site, Propath UK Ltd, Willow Court, Netherwood Road, GB - Hereford HR2 6JU.

Statistics:

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

Clinical signs:

no effects observed

Mortality:

no mortality observed

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):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: No effects observed

Critical effects observed:

not specified

Mortality

	C (0 mg/kg)	LD (250 mg/kg)	MD (500 mg/kg)	HD (1000 mg/kg)
Total number of animals examined: males	10	10	10	10
Total number of animals examined: females	10	10	10	10
No mortality was observed in any male from various treatment and control group during the entire period of the study. One female animal from low dose was found dead on gestation day 15 due to gavaging error (not attributed to test item).

 

 

Clinical Observations – Male

Clinical Finding	C (0 mg/kg)	LD (250 mg/kg)	MD (500 mg/kg)	HD (1000 mg/kg)
Total number of animals examined	10	10	10	10
No clinical signs were observed in any male from various treatment and control group during the entire period of the study.

 

Clinical Observations – Female

Clinical Finding	C (0 mg/kg)	LD (250 mg/kg)	MD (500 mg/kg)	HD (1000 mg/kg)
Total number of animals examined	10	10	10	10
No clinical signs were observed in any female from various treatment and control group during the entire period of the study.

 

Reproductive Indices

Index	Group
		C (0 mg/kg)	LD (250 mg/kg)	MD (500 mg/kg)	HD (1000 mg/kg)
Copulation Index	(%)	100	100	100	100
Fertility Index	(%)	90	100	80	100
Delivery Index	(%)	100	100	100	100
Viability Index	Mean	100.00	100.00	100.00	97.00

Copulation Index (%)= (No. of rats copulated /No.of pairs)X 100

Fertility Index (%)= (No. of Females Pregant/No.of females copulated)x 100

Delivery Index(%)= (No. of dams with live newborns/ No.of pregnant dams)X 100

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

No. of live offspring at day 4/ No.of live offspring at birth)x 100

 

Macroscopic Findings - Male

Findings (External/Internal)	C (0 g/kg)	LD (250 mg/kg)	MD (500 mg/kg)	HD (1000 mg/kg)
Total number of animals examined	10	10	10	10
Left seminal vesicle diminished	1	0	0	0
Epididymidis-yellowish, whitish deposition (bilateral)	1	0	1	0
Right epididymis-yellowish, whitish deposition	0	1	1	0
Lung redish discolouration	0	0	1	0
Left Epididymidis-yellowish, whitish deposition	0	0	1	1
Right seminal vesicle with white spots( 1-2mm)	0	0	0	1

 

Macroscopic Findings- Female

Findings (External/Internal)	C (0 g/kg)	LD (250 mg/kg)	MD (500 mg/kg)	HD (1000 mg/kg)
Total number of animals examined	10	10	10	10
Lymph nodes axillary: left slightly enlarged; right red discolouration at the margin	1	0	0	0
Lung-residuals of the test item	0	2	1	2
Lung-bloody infiltrated,foam	0	1	0	0
Kidney- small cyst on right kidney.	0	0	1	0
Thymus- slightly reduced in size	0	0	2	0
Lung-slight bloody, red spots	0	0	1	0
oesophagos-residuals of the test item	0	0	1	0
Axillary lymphnode-left slightly enlarged	0	0	0	1
Lung- bloody, red discoloured	0	0	0	2

 

 

Tabular Study Summary

OBSERVATIONS					Group
Dosage (units).		C (0 mg/kg)	LD (250 mg/kg)				MD (500 mg/kg)			HD (1000 mg/kg)
Pairs started (N)		10	10				10			10
Females showing evidence of copulation (N)		10	10				10			10
Females achieving pregnancy (N)		9	10				8			10
Conceiving days 1 - 5 (N)		9	10				10			10
Conceiving days 6 - . . .(1) (N)		1	0				0			0
Pregnancy = 21 days or below (N)		0	0				0			0
Pregnancy = 22 days (N)		6	5				7			7
Pregnancy ³ 23 days (N)		3	4				1			3
Dams with live young born (N)		9	9				8			10
Dams with live young at day 4 pp (N)		9	9				8			10
Corpora lutea/dam (mean)		12.56	12.67				12.88			12.30
Implants/dam (mean)		11.33	10.67				11.00			10.70
Live pups/dam at birth (mean)		10.89	10.11				10.63			10.20
Live pups/dam at day 4 (mean)		10.89	10.11				10.63			9.90
Sex ratio (m/f) at birth (mean)		1.78	0.85				0.98			1.16
Sex ratio (m/f) at day 4 (mean)		1.78	0.85				0.98			1.12
Litter weight at birth (mean)		65.10	62.70				62.64			59.61
Litter weight at day 4 (mean)		111.18	107.44				108.90			101.48
Pup weight at birth (mean)		6.02	6.26				5.91			5.87
Pup weight at day 4 (mean)		10.19	10.74				10.42			10.30
ABNORMAL PUPS
Dams with 0		8	8				6			8
Dams with 1		1	0				2			1
Dams with 2		0	1				0			1
Dosage (units).		C (0 mg/kg)	LD (250 mg/kg)				MD (500 mg/kg)			HD (1000 mg/kg)
LOSS OF OFFSPRING
Pre-implantation (corpora lutea minus implantations)
Females with 0	4			1				3		3
Females with 1	2			2				2		1
Females with 2	1			3				2		4
Females with 3 and more	2			3				1		2
Pre-natal (implantations minus live births)
Females with 0	6			5				5	6
Females with 1	2			3				3	3
Females with 2	1			1				0	1
Females with 3	0			0				0	0
Post-natal (live births minus alive at post natal day 4)
Females with 0	9			9		8			8
Females with 1	0			0		0			1
Females with 2	0			0		0			1
Females with 3	0			0		0			0

Conclusions:

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.6, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

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

Reason / purpose for cross-reference:

reference to other study

Remarks:

14-day inhalation dose rangefinder study

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

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

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

Dose / conc.:

1.5 mg/m³ air (analytical)

Remarks:

target aerosol concentration = 1.5 mg/m3

Dose / conc.:

5.99 mg/m³ air (analytical)

Remarks:

target aerosol concentration = 6 mg/m3

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.

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.

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).

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)

Key result

Critical effects observed:

no

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

24 mg/m³

Study duration:

subchronic

Experimental exposure time per week (hours/week):

30

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex IX, 8.6, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

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

Reason / purpose for cross-reference:

reference to other study

Remarks:

14-day inhalation dose rangefinder study

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

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

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

Dose / conc.:

1.5 mg/m³ air (analytical)

Remarks:

target aerosol concentration = 1.5 mg/m3

Dose / conc.:

5.99 mg/m³ air (analytical)

Remarks:

target aerosol concentration = 6 mg/m3

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.

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.

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).

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)

Key result

Critical effects observed:

no

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

24 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex IX, 8.6, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

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

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

A sub-chronic inhalation toxicity study has also been performed to investigate the adverse and adaptive effects of diniobium pentaoxide following a 90-day nose-only inhalation study in the rat (+ up to 90 days post-exposure observation).

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

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

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

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

In conclusion, based on the absence of adverse effects observed in the study, the NOAEL in male and females after sub-acute exposure was determined to be 1000 mg/kg bw/day.

Sub-chronic repeated dose inhalation toxicity study in rat

A 90-day inhalation study was conducted in rats using nose-only exposure, the study was conducted according to OECD 413 and in compliance with GLP.

The objectives of this study were as follows:

To investigate the adverse and adaptive effects of diniobium pentaoxide following a 90-day nose-only inhalation study in the rat (+ up to 90 days post-exposure observation).

To investigate the lung toxicity potential of diniobium pentaoxide using clinical observations, haematology and clinical chemistry, gross pathology, histopathology, bronchoalveolar lavage (BAL) fluid analysis and lung burden analysis (the latter only storage of samples) as endpoints.

To establish exposure-dose-response relationships of the inhaled test item in rats after sub-chronic exposure and to derive NOAEC and LOAEC values.

To check reversibility and adaptive effects of the test item in the respiratory tract as compared to historical data with a positive particle control, e.g. Quartz DQ12.

One-hundred male and sixty female Wistar rats [strain Crl:WI (Han)] were used for this study and allocated to four groups each: Clean air control, diniobium pentaoxide low (1.5 mg/m³), diniobium pentaoxide mid (6 mg/m³) and diniobium pentaoxide high (24 mg/m³). The dose levels were selected based on the findings in a dose range finding study, which is included as a supporting study.

The target aerosol concentrations of 1.5, 6 and 24 mg/m³ dinobium pentaoxide were achieved exactly, i.e. to 100% in each group.

All animals survived the test period and were euthanized at scheduled dates. Effects indicating systemic toxicity were not observed. Sex-specific differences were not detected.

The observations can be summarized as follows:

Body weight development, food and water consumption, haematology and clinical chemistry parameters were unaffected in both sexes, no relevant statistically significant changes were observed as compared to concurrent controls.

In both sexes, no relevant statistically significant changes as compared to concurrent controls were observed.

For the bronchoalveolar lavage fluid (BALF) analysis, the following was observed:

At Days 1 and 90 post-exposure, no relevant statistically significant increases of polymorphonuclear neutrophils (PMN) 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 post-exposure, 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 absence 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.

Histopathological evaluation revealed no adverse findings. 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 lung burden analysis was conducted due to the unremarkable findings of the BAL analysis and the histopathological examinations.

Overall, under the conditions of the study, a NOAEC of ≥ 24 mg/m3 was derived for both sexes. The test item diniobium pentaoxide was therefore considered to be a typical inert dust.

Justification for classification or non-classification

The available data on repeated dose toxicity does not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and is therefore conclusive but not sufficient for classification.