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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
fertility, other
Remarks:
male reproductive system
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
Assessment of the Effects Following Subchronic Dosing with Sodium Tungstate on Male Reproductive System in Wistar Rats
Author:
Pandey G. et al.
Year:
2011
Bibliographic source:
Recent Research in Science and Technology 3(11), 55-60

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Sodium tungstate was orally administrated to male Wistar rats at a dose level of 50 mg/kg bw/day for 60 days to investigate the effects of sodium tungstate on male reproductive system
GLP compliance:
not specified
Remarks:
it was not identified by the authors if the study was GLP compliant
Limit test:
no

Test material

Constituent 1
Reference substance name:
Reference substance 001
EC Number:
600-275-2
Cas Number:
10213-10-2
Specific details on test material used for the study:
TEST MATERIAL FORM
- solid: particulate/powder

DETAILS ON TEST MATERIAL
- Name of test material (as cited in study report): Sodium tungstate
- Other: purchased from Merck India Ltd., Mumbai, India

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 170 - 200 g
- Housing: standard rat cages
- Diet (e.g. ad libitum): standard laboratory chow, ad libitum
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25 +/- 3
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
distilled
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The daily dose of the chemical was dissolved in 0.5 mL of distilled water
Details on mating procedure:
Fertility Test:
Male rats were introduced to parous females, 170-200 g (male:female ratio 1:2) at 55 days of treatment. The successful mating was confirmed in the forthcoming mornings from 56 to 61 days by vaginal plug and spermatozoa in the vaginal smear. The inseminated females were seperated and allowed to deliver at term. The number of pups delivered and their characterictis were noted.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
60 days
Frequency of treatment:
daily
Doses / concentrations
Dose / conc.:
50 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Group A: control rats received 0.5 mL/day of distilled water
Group B: rats treated with Sodium tungstate dihydrate
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: before the beginning of treatment, at weekly intervals, and at the end of treatment

FOOD CONSUMPTION: Yes
Food consumption was monitored daily, water containers were checked each day and protected from light
Sperm parameters (parental animals):
Parameters examined in [P] male parental generation: The testes, epididymides, vas deferens, seminal vesicles and ventral prostate, and adrenal removed, cleared of the adhering connective tissue and weighed. Testes and epididymis were assayed for protein, sialic acid, glycogen and cholesterol. Fructose in seminal vesicle was also estimated. Blood samples were collected for estimations of serum testosterone, follicle stimulating hormone and Luteinizing Hormone. Spermatozoa from cauda epididymis were counted. Total sperm Number and cauda epididymal sperm counts were determined. For sperm motility, one drop of evenly mixed sample was applied to a glass slide under a cover glass, percent motility was determined by counting both motile and immotile spermatozoa per unit area. Mean seminiferous tubular diameter was determined and diameter of Leydig cells nuclei were measured.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring: The number of pups delivered and their characteristics were noted following [WHO, 1983. Protocol MB-50, A Method for Examining the Effect of the Plant Extracts Administered Orally on the Fertility of Male Rats 9914E. World Health Organization, Geneva].
Postmortem examinations (parental animals):
SACRIFICE / GROSS PATHOLOGY
- Male animals: All surviving animals were weighed and autopsied under light ether anesthesia 24h after the last dose of treatment.

HISTOPATHOLOGY:
Testes were fixed in Bouin`s fluid. Paraffin section were cut (5 µm) and stained with hematoxylin and eosin. Mean seminiferous tubular diameter was determined by measuring 100 round sections of seminiferous tubule with the help of ocular micrometer. Diameter of Leydig cells nuclei were measured at x 800.

ORGAN WEIGHTS
The testes, epididymides, vas deferens, seminal vesicles and ventral prostate, kidney, adrenal and liver were removed, cleared of the adhering connective tissue and weighed.

TISSUE BIOCHEMISTRY:
Testes, epididymis and accessory sex organs were frozen at -20 °C for the biochemical estimations. Testes and epididymis were assayed for protein, sialic acid, glycogen and cholesterol. Fructose in seminal vesicle was also estimated.

RADIOIMMUNOASSAY:
Blood samples were also collected for estimations of serum testosterone by radioimmunoassay. Follicle-stimulating hormone and Luteinizing hormone
Statistics:
Data are expressed as mean ± S.E.M. One-way analysis of variance (StatPlus, 2007) was used for statistical comparison.
Reproductive indices:
Fertility of male rats was assesed by the incidence of pregnancy in females

Epididymal sperm concentration and motility:
Spermatozoa from cauda epididymis were counted. One hundred milligram of the epididymis was finely minced with anatomical scissors in 1 mL of physiological saline, placed in a rocker for 10 min then allowed to sit at room temerpature for 2 min. Total sperm number was determined by using a hemocytometer. For sperm motility, one drop of evenly mixed sample was applied to a glass side under a cover glass. The percent motility was determined by counting both motile and immotile spermatozoa per unit area.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicity were observed
Mortality:
no mortality observed
Description (incidence):
No deaths were observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The growth and general appearance of the test item treated animals was normal throughout the experiment. The test item did not cause any significant adverse effect on the body weight of treated rats.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Treatment with the test item produce non significant depletion in protein, glycogen and sialic acid content in testis and epididymis and fructose level in seminal vesicle. Cholesterol level of testes and epididymides was non significantly increased.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Treated groups were essentially normal, containing only incidental microfocal lesions of seminiferous or epididymal tubule. Spermatogenesis does not appear to be affected. No change in diameter of seminiferous tubular and number of Leydig cells. There were no histopathological changes ofound in cauda epididymis of treated rats.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Non-significant reduced sperm count and sperm motility in cauda epididymis
Reproductive performance:
no effects observed
Description (incidence and severity):
No significant change in the fertility of male rats as assessed by the incidence of pregnancy in females.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no adverse effects observed

Results: F1 generation

Effect levels (F1)

Remarks on result:
other: this study was designed to assess the effects of the treatment on the male reproductive organ

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Table 1: Effect of the test item treatment on the body and organ weight of male albino rats

Body weight (g) Reproductive organs weight (mg/100 g body weight) Vital organ weight (mg/100 bd.wt)
Treatment Initial Final Testes Epididymides Ventral prostate Seminal vesicle Vas deferens Kidney Adrenal Liver
Group A, control (vehicle-treated) 173.75 ±5.66 201.87 ±4.33 1174.50 ±43.84 497.51 ±62.56  283.08 ±16.63 401.31 ±14.95 104.58 ±4.75  590.97 ±21.36 22.43 ± 1.02 3229.53 ± 72.25
Group B, 50 mg/kg body weight/day for 60 days 172.5 ±2.84 221.62 ±3.54 1089.08ns ±19.4 (p<0.125) 474.03ns ±10.32 (p<0.2119) 267.57ns ±10.81 (p<0.5036) 395.59ns ±8.36 (p<0.759) 98.34ns ±2.61 (p<0.271) 550.17ns ±11.61 (p<0.137) 19.92 ns ±1.12 (p<0.189) 3183.75ns ± 71.88 (p<0.669)

Data are expressed as Mean ± SEM of eight animals. ANOVA analysis of variance; Groups B was compared with Group A, a Highly

significant (p≤ 0.0001) ; b Significant (P≤.001); ns non significant

Table 2: Effect of the test item treatment on tissue biochemistry of male albino rats

Cholesterol (mg/g) Sialic acid (mg/g) Glycogen (mg/g) Total protein (mg/g) Fructose
Treatment Testes Epididymides Testes Epididymides Testes Epididymides Testes Epididymides Seminal vesicle
Group A, control (vehicle-treated) 4.98 ± 0.30 4.72 ± 0.17 4.63 ± 0.16 4.51 ± 0.16 3.34 ± 0.14 3.96 ± 0.13 194.37 ± 6.81 198.12 ± 8.59 3.12 ± 0.21
Group B, 50 mg/kg body weight/day for 60 days 5.06ns ± 0.21 (p<0.801) 5.03ns ± 0.04 (p<0.110) 4.45ns ± 0.15 (p<0.446) 4.35ns ± 0.20 (p<0.5461) 3.13ns ± 0.15 (p<0.178) 3.39ns ± 0.17 (p<0.127) 192.63ns ± 5.57 (p<0.855) 197.37ns ± 5.07 (p<0.941) 3.20ns ± 0.12 (p<0.737)

Data are expressed as Mean ± SEM of eight animals. ANOVA analysis of variance; Groups B was compared with Group A, highly significant (p≤ 0.0001) ; b

Significant (P≤.001); ns non significant

Table 3: Effect of Sodium tungstate treatment on serum testosterone, serum FSH, serum LH, cauda epididymis sperm analysis, fertility test and

morphometry of male albino rats

Treatment Serum testosterone (ng/L) Serum FSH (ng/mL) Serum LH (ng/ml) Sperm count (million/mm3) Motility (%) Cauda epididymis Fertility test (%) Seminiferous tubule diameter (µm) Leydig cell nuclei diameter (µm)
Group A, control (vehicle-treated) 4.06 ± 0.11 112.87 ± 1.39  4.04 ±0.057 44.14 ±0.628 63.33 ±1.27 93.75 258.12 ±4.79 6.07 ±0.089
Group B, 50 mg/kg body weight/day for 60 days 3.95ns ±0.10 (p<0.4745) 108.25ns ±1.164 (p<0.381)  3.93ns ±0.063 (p<0.212) 42.95ns ±0.66 (p<0.2094) 60.31ns ±1.69 (p<0.172)  87.5 248.12ns ±4.29 (p<0.1343) 5.93ns ±0.13 (p<0.383)

Data are expressed as Mean ± SEM of eight animals. ANOVA analysis of variance; Groups B was compared with

Group A, a Highly significant (p≤0.0001); b Significant (P≤.001); ns non significant

Applicant's summary and conclusion

Conclusions:
In conclusion, the test item (50 mg/kg bw/day) did not cause any severe histological and biochemical alteration in male reproductive system and thus the NOAEL can be considered to be 50 mg/kg bw/day.
Executive summary:

In a subchronic repeated dose toxicity study with focus on the male reproductive system, Sodium tungstate dihydrate was administered daily to male Wistar rats by oral gavage at dose levels of 0 and 50 mg/kg bw/day for a total of 60 days. Treated males were mated with untreated females after 55 days of treatment, which were then allowed to deliver their offspring at term.

No deaths or any other clinical signs of toxicity were observed. No effects were seen on body or organ weight. Although there was a non-significant reduction in sperm count and sperm motility in cauda epididymis, there was no significant change in the fertility of male rats. Treated rats showed essentially normal histopathology. There was a non-significant reduction in protein, glycogen and Sialic acid content of testes and epididymides, in fructose levels in the seminal vesicle, and in serum testosterone levels, FSH and LH levels. The cholesterol content of testes and epididymides was non-significantly increased after the administration of sodium tungstate. Overall, results in the study do not indicate that Sodium tungstate dehydrate exposure resulted in direct male reproductive toxicity in the rat. Based on the results from this study the NOAEL can be considered to be 50 mg/kg bw/day.