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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
reproductive toxicity, other
Remarks:
repeated dose 28-days oral toxicity study
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Justification for type of information:
Data from an experimental study report.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
other: OECD guideline No. 407
Principles of method if other than guideline:
Repeated Dose 28-Days Oral Toxicity Study of test chemical in Sprague Dawley (SD) rats to determine toxicity to reproductive organ.
GLP compliance:
not specified
Limit test:
no
Justification for study design:
No data available

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report):Zinc Distearate
- Molecular formula :C18H36O2.1/2Zn
- Molecular weight :632.3476 g/mol
- Substance type:Organic
- Physical state:Solid (Powder)
- Impurities (identity and concentrations):0.14 %

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
No data available
Sex:
male/female
Details on test animals and environmental conditions:
Details on test animal
TEST ANIMALS
- Source: Central Animal Facility (CAF), NIPER, Sector-67, S.A.S. Nagar, 160 062, Punjab, India.

- Age at study initiation: 7 to 8 weeks old

- Weight at study initiation: Male: 205.78-247.70 g, Female : 185.58-235.40 g

- Fasting period before study: No data available
- Housing: Animals were housed in sterilized solid bottom polypropylene cages with stainless steel grill tops with facilities for food, water bottles and bedding of clean paddy husk. The cages were suspended on stainless steel racks. Animals were identified by assigning a unique identification (ID) number written on the tail, also specified on individual cage tag.

- Diet (e.g. ad libitum): Standard laboratory sterile extruded pelleted rodent feed (Batch No. 0001642169) manufactured by Provimi Animal Nutrition India Pvt. Ltd., Bangalore, ad libitum.

- Water (e.g. ad libitum): Potable tap water filtered through Reviva Reverse Osmosis System (water filter cum purifier) of Eureka Forbes in polypropylene bottles with stainless steel sipper tubes. ad libitum.

- Acclimation period: Five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%):30-70 %,
- Air changes (per hr): 25 ± 5 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Details on oral exposure
PREPARATION OF DOSING SOLUTIONS: Dose was freshly prepared prior to dosing on each day. Corn oil was used as a vehicle for this study.Test chemical was administered to each rat at the dose levels of 250, 500 and 1000 mg/kg in the dose volume of 5 ml/kg body weight. The chemical was weighed on a weighing balance. Then, it was transferred to calibrated falcon tube. Some quantity of the corn oil was added initially and vortexed. The sufficient quantity of vehicle was added to make up the required volume of dose.


DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): No data available

- Storage temperature of food: No data available

VEHICLE
- Justification for use and choice of vehicle (if other than water): Chemical was insoluble in water, the corn oil was used as vehicle to deliver the desired dose levels as it is also recommended in the toxicological evaluation guidelines.

- Concentration in vehicle: 0, 250, 500 and 1000 mg/kg/day
- Amount of vehicle (if gavage): 5 ml of corn oil
- Lot/batch no. (if required): Batch no. 51
- Purity: No data available
Details on mating procedure:
No data available
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The tet chemical (250, 500 and 1000 mg) was weighed on balance and dissolved in 5 ml of corn oil. An aliquot (5µl) was taken from three different layers of the dose preparations and was mixed in toluene to make the final volume 1ml. The concentration of the substence in each layer was calculated using the calibration curve of standard (5 mg / ml toluene) by UV - Visible spectroscopy using Flex Station at 600 nm.
Duration of treatment / exposure:
28 days.
Frequency of treatment:
Daily
Details on study schedule:
No data available
Doses / concentrations
Remarks:
0, 250, 500 and 1000 mg/kg/body weight/day
No. of animals per sex per dose:
Total: 56
0 mg/kg/day: 7 male, 7 female
250 mg/kg/day: 7 male, 7 female
500 mg/kg/day: 7 male, 7 female
1000 mg/kg/day: 7 male, 7 female
Control animals:
yes, concurrent vehicle
Details on study design:
No data available
Positive control:
No data available

Examinations

Parental animals: Observations and examinations:
Observations and examinations performed & frequency
CAGE SIDE OBSERVATIONS: Yes
- Time schedule : Twice daily (morning and evening)
- Cage side observations checked in table [No.?] were included. : Viaibility or moribund

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily for behavioral changes or reaction to treatment and detailed clinical signs were recorded weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: On day 1, 8, 15, 22, 28, and day 29 (before schedule sacrifice)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data available
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data available
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data available

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data available

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Weekly once during 28 days of treatment.

Oestrous cyclicity (parental animals):
No data available
Sperm parameters (parental animals):
No data available
Litter observations:
No data available
Postmortem examinations (parental animals):
Sacrifice and pathology
GROSS PATHOLOGY: Yes
All animals were sacrificed by CO2 asphyxiation. Complete necropsy was carried out on all the animals to score the gross lesions. Tissues were collected from all animals and preserved in 10% formal saline. However, testes, ovaries and uterus were first fixed in Bouin’s fixative for short duration then transferred to 10% formal saline.


HISTOPATHOLOGY: Yes
The required tissues for histology slide preparations were embedded in paraffin wax, five micrometers tissue sections were cut and stained with haematoxylin and eosin and examined for histopathological changes.

Organ examined: Brain, Stomach, Large intestine Small intestine, Liver, Kidneys, Adrenal gland(s), Spleen, Heart, Thymus, Lungs, Testis / Ovary, Uterus, Sciatic nerve Lymph nodes, Peripheral nerve (Sciatic) and Bone marrow were examined.
Postmortem examinations (offspring):
No data available
Statistics:
Statistical analysis was carried out by using Microsoft Excel and IBM SPSS statistics version-20.0. All analyses and comparisons were evaluated at the 5 % level, statistically significant differences (p ≤0.05) indicated by appropriate notation. The focus was to examine the mean differences and their significance between control and low dose group, control and mid dose group and control and high dose group. The statistical decision was taken by preparing the univariant GLM MODEL procedure was used to check the significance between above mentioned groups. For multiple comparisons Turkey’s HSD test was applied.
Reproductive indices:
No data available
Offspring viability indices:
No data available

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Soft stool, nasal discharge was observed in all male and female treated animals.

When treated with 250 mg/kg, crust around nostrils in male and perineum soiled with fecal matter in female were observed.

In 1000 mg/kg/dose, red crust around nostrils, perineum soiled and soft feces were observed in female rats.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
No mortality was observed in treated animals.

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
When treated with 500 and 1000 mg/kg/day, In male rat, significant increase were observed. In female rats, significant increase in body weight were observed in 1000 mg/kg/day as compare to control.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No change was observed in food consumption of male and female rat of treated groups.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No change was observed among all treated male rat but in female rat significant decreased was observed on 4th week of 1000 mg/kg/day dose treatment
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No abnormalities were observed in all treated dose groups rat.
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In male rats, when treated with 1000 mg/kg/day testosterone, sodium, total proteins and total cholesterol level were significantly increased and SGOT, SGPT were significantly reduced in treated rats.

Creatinine level was significantly reduced in 500 mg/kg/day treated male rats.

Potassium level was significantly increased in 250 mg/kg/day treated male rat.

When treaed with 1000 mg/kg/day, In female rats, significant reduction were observed in sodium, albumin and glucose level and Total Bile Acids, Potassium and SGPT were significant ly increased as compared to control.

When treated with 500 mg/kg/day, Potassium and SGPT significantly increased and glucose, blood urea nitrogen (BUN) and creatinine were significantly decreased in female treated rat.

When treated with 250 mg/kg/day, Potassium, SGPT and estrogen was significantly increased and SGOT were significantly decreased in female treated rat.
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
In male rat treated with 1000 mg/kg/day, absolute weight of brine and heart were increased significantly in treated rat. Relative weight of brine, liver, kidneys, thymus, testes, heart and epididymides were decreased significantly in 1000 mg/kg/day treated male rat. When treated with 500 mg/kg/day, absolute weight of brine and heart were increased significantly while relative weight of liver and epididymides were decreased significantly in treated male rat.When treated with 250 mg/kg/day, absolute and relative weight of brine and absolute weight of spleen were increased significantly Absolute and relative weight of adrenals, absolute weight of thymus and epdidymides and relative weight of liver and kidneys were decreased in 250 mg/kg/day treated male rat. In female rat, treated with 1000 mg/kg/day absolute and relative weight of thymus and spleen and absolute weight of brine and heart were increased significantly in treated rat.Absolute and relative weight of ovaries and relative weight of liver were decreased significantly in treated rat. When treated with 500 mg/kg/day, absolute and relative weight of uterus increased and ovaries decreased significantly while relative weight of kidneys increased significantly in treated rat. When treated with 250 mg/kg/day, absolute and relative weight of adrenals and ovaries and relative weight of brine decreased significantly in female treated rats. However, the biological significance of these findings are not related to test chemical.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Extra marks and fatty mass on heart were observed in 500 mg/kg/day dose and dark colored spot on lung and fat deposition around small and large intestine in 1000 mg/kg/day treated male rat. In female rat, fat deposition around small and large intestine and tissue growth around heart was observed in 250 mg/kg/day dose, tumor like development (approximately 1 cm) in hind limb region (RHS), spotted lung (Red) and fat deposition in abdominal region was observed in 500 and 1000 mg/kg/day dose spotted lung, fat deposition in abdominal region and cavity was observed in treated rat as compare to control.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Minimal increased were observed in eosinophils of small intestine, collapsed lung, minimal focal fatty change in liver were observed in 1000 mg/kg/day male rats. In female, minimal focal fatty change in liver, minimal reactive spleen and minimal increased in eosinophils of small intestine and mild changes were observed in liver with focal fatty change and reactive spleen was observed in 1000 mg/kg/day dose animals.However, the biological significance of these findings are not related to test chemical.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
not specified

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
ophthalmological examination
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: No toxic effects on reproductive organ was observed

Target system / organ toxicity (P0)

Critical effects observed:
not specified
System:
other: not specified
Organ:
not specified
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not specified

Effect levels (F1)

Dose descriptor:
other: not specified
Generation:
other: not specified
Based on:
not specified
Sex:
not specified
Basis for effect level:
other: not specified
Remarks on result:
not measured/tested

Overall reproductive toxicity

Reproductive effects observed:
not specified
Treatment related:
not specified
Relation to other toxic effects:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Any other information on results incl. tables

Table: Summary of Body Weights

Treatment group

Time (Days)

1

8

15

22

28

Terminal day

Males   

Control

217.79

247.35

269.99

290.96

304.89

300.87

250 mg/Kg

224.64

254.35

279.94

305.36

318.22

313.79

500 mg/Kg

228.39

256.96

282.73

312.63

327.08

322.00

1000 mg/Kg

233.18*

261.44*

284.33*

312.10*

322.86

319.86

Females

Control

194.50

206.76

217.21

231.38

234.65

231.10

250 mg/Kg

195.28

206.89

215.12

225.92

232.35

229.71

500 mg/Kg

199.97

214.31

224.03

235.82

241.54

238.41

1000 mg/Kg

217.27*

233.49*

241.12*

254.07*

261.27*

257.44*

 * Significant at p < 0.05 in comparison to control group

Table: Summary of Histopathology findings

Gross findings

Dose groups

Males

Females

G1

G2

G3

G4

G5

G6

G7

G8

Incidence (No. of Animals with Findings/ No. of Animals Examined)        

 

 

 

1/7

-

-

3/7

2/7

-

-

3/7

Lung

 

 

Lung Collapsed

-

-

-

1/7

-

-

-

-

Liver

 

 

Focal Fatty change

-

-

-

3/7

1/7

-

-

1/7

Small intestine

 

 

Excess Lymphocyetes

1/7

-

-

-

-

-

-

1/7

Spleen

 

 

Reactive

-

-

-

-

1/7

-

-

1/7

Applicant's summary and conclusion

Conclusions:
NOAEL was considered to be 1000 mg/kg/bw/ day in male and female rats when exposed to test chemical for 28 days.
Executive summary:

In 28 days repeated dose toxicity study, the effect of test chemical was evaluated in male and female Sprague Dawley rats. The test chemical was administered by oral gavage in the concentration of 0, 250, 500 and 1000 mg/kg/ body weight/day. The results showed no effect on mortality, no changes were in food consumption and ophthalmology. Changes were observed in clinical signs like soft stool, nasal discharge, red crust around nostrils, perineum soiled with fecal matter. Significant decreased were observed in water consumption and locomotor activity of female rat in1000 mg/kg dose group. Body weight was increased significantly in male and female rats. Significant changes were observed in the level of testosterone, sodium, total proteins, total cholesterol, SGOT, SGPT, albumin, Blood urea nitrogen (BUN) and Creatinine when treated with 500 and 1000 mg/kg/day. Significant changes were observed in absolute and relative weight of brain, adrenals, spleen, thymus, epididymides, heart, kidneys, ovaries, uterus and liver in 500 and 1000 mg/kg/day. In addition, minimal to mild gross pathological and histopathological changes were observed in liver, spleen and intestine. However, the biological significance of these findings are not related to test chemical. Therefore, NOAEL was considered to be 1000 mg/kg/day when Sprague-Dawley rat exposed to test chemical orally.