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Repeated dose toxicity: oral

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Administrative data

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:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
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)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

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.

Administration / exposure

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
LD: 250 mg/kg bw
MD: 500 mg/kg bw
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 bsiss. 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
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
Control: 0 mg/kg bw
Basis:
nominal in water
Remarks:
Doses / Concentrations:
LD: 250 mg/kg bw
Basis:
nominal in water
Remarks:
Doses / Concentrations:
MD: 500 mg/kg bw
Basis:
nominal in water
Remarks:
Doses / Concentrations:
HD: 1000 mg/kg bw
Basis:
nominal in water
No. of animals per sex per dose:
10
Control animals:
yes
Positive control:
no

Examinations

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 includeed 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 inflatation 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.

Results and discussion

Results of examinations

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

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw (total dose)
Sex:
male/female
Basis for effect level:
other: No effects observed

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

Conclusions:
In conclusion, the repeated dose administration of Diniobium Pentoxide (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 body weight revealed no major toxicological findings.
Based on the data generated from this combined repeated dose toxicity and reproduction/ developmental toxicity screening test with Diniobium Pentoxide, the no observed adverse effect level (NOAEL) is believed to be 1000 mg/kg body weight in males and females.