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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: 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

Constituent 1
Chemical structure
Reference substance name:
Diniobium pentaoxide
EC Number:
215-213-6
EC Name:
Diniobium pentaoxide
Cas Number:
1313-96-8
Molecular formula:
Nb2O5
IUPAC Name:
diniobium(5+) pentaoxidandiide

Test 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 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

Parental animals: Observations and examinations:
General clinical observations were made twice a day except during weekend and holidays where observations were made only once, approximately at the same time each day and considering the peak period of anticipated effects after dosing.Pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality were recorded.
Once before the first exposure, and once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoe, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), and piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypies, difficult or prolonged parturition or bizarre behaviour was recorded.
Sensory reactivity to different modalities, grip strength and motor activity assessments and other behaviour observations were conducted on five randomly selected males and females from each group. Observations were 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.
Litter observations:
Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.
Postmortem examinations (parental animals):
All survived male animals were sacrificed on day 29 and 30. Non pregnant females were sacrificed on the respective day 26 after the sperm positive vaginal smear as an evidence of mating. Lactatating females along with pups were sacrificed on respective post natal day 4.
Statistics:
Parameters like body weight change and food consumption was calculated for each animal as the difference in weight measured from one week to the next. The relative organ weights were calculated in relation to the body weight (measured at necropsy) and presented as percentage.
All results are reported in tabular form (summarized in mean or summary tables and listed in individual data tables). Mean body weights are also presented as figures.
Analytical results and histopathological findings are presented in separate phase-reports attached to this report.
For statistical analysis one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test was carried out to reveal any differences between control and test groups. Statistical analysis performed with GraphPad Prism V.x software (p<0.05 was considered as statistical significant).
In the evaluation of laboratory parameters, all values within a range of the mean value ± the two fold standard deviation (x ± 2s) are considered to be "normal“ values within a "normal“ population.
Reproductive indices:
calculated

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

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

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

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

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

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

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

Effect levels (P0)

Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: No adverse and treatment-related effects were observed

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

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

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: No adverse and treatment-related effects were observed.

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

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.