Zinc distearate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Principles of method if other than guideline:

Repeated Dose 28-Days Oral Toxicity Study of test chemcial in Sprague Dawley (SD) rats was conducted to evaluate its toxic nature.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Zinc distearate

EC Number:

209-151-9

EC Name:

Zinc distearate

Cas Number:

557-05-1

Molecular formula:

C18H36O2.1/2Zn

IUPAC Name:

zinc distearate

Details on test material:

- Name of test material: Zinc Distearate (ZDS)
- Molecular formula: C18H36O2.1/2Zn
- Molecular weight: 632.3476 g/mol
- Substance type: Organic
- Physical state: Solid (Free flowing white Powder)
- Impurities (identity and concentrations): 0.14 %

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

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 no more than 4/sex 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
- Quarantine period: 3 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 Deg 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

IN-LIFE DATES: From: 20.05.2014
To: 27.06.2014

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

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 chemcial was administered to each rat at the dose levels of 0, 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/Kg/bw of corn oil
- Lot/batch no. (if required): Batch no. 51
- Purity: 99.86%

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Substance (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 substance in each layer was calculated using the calibration curve of standard (5 mg ZDS / ml toluene) by UV - Visible spectroscopy using Flex Station at 600 nm.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily for 28 consecutive days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

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:

- Dose selection rationale: Doses were selected on the basis of information provided by the sponsor. The three dose levels of test chemical selected for this study were 250, 500 and 1000 mg/kg body weights of test animals.
- Rationale for animal assignment (if not random): Animals were randomized by body weight and sex.
- Rationale for selecting satellite groups: No data available
- Post-exposure recovery period in satellite groups: No data available
- Section schedule rationale (if not random): No data available

Positive control:

No data

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule : Twice daily (morning and evening) for mortality and daily for cage side clinical examinations
- Cage side observations checked in table [No.?] were included. : Visibility or moribund and mortality, to characterize onset and duration of clinical signs

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily for behavioral changes or reaction to treatment and detailed clinical signs were recorded weekly. Detailed physical and clinical observations were conducted before the first exposure of test item and at least once a week thereafter till the end of the study. The clinical signs were recorded daily 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): Yes, Food consumption was recorded weekly once during the entire course of treatment.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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. The measured volume of water was offered in each cage and the left volume was measured after 24 hrs and finally the water intake per rat was calculated.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: In fourth week of treatment.
- Dose groups that were examined: All 56 animals were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood were collected prior to necropsy.
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes, overnight fasting before necropsy
- How many animals: All 56 animals were examined.
- Parameters checked in table [No.?] were examined.: Haemoglobin (Hb), RBC Count, Total and differential leucocyte count (TLC / DLC), Haematocrit (Hct / PCV), Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC) and Platelet count were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood were collected prior to necropsy.
- Animals fasted: Yes, overnight fasting before necropsy
- How many animals: All 56 animals were examined.
- Parameters checked in table [No.?] were examined.: Sodium and Potassium, Glucose, Total Cholesterol, Blood Urea Creatinine, Total Protein, Albumin, SGPT (Serum glutamic pyruvic transaminase) /ALT, SGOT (Serum glutamic oxaloacetic transaminase) /AST, Hormones analysis (testosterone and estrogen) and Total Bile Acids were examined.

URINALYSIS: No data available
- Time schedule for collection of urine: No data available
- Metabolism cages used for collection of urine: No data available
- Animals fasted: No data available
- Parameters checked in table [No.?] were examined.: No data available

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: In the fourth exposure week
- Dose groups that were examined: All 56 rats were examined
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Yes, locomotor activity was tested on fourth exposure week of treatement. Each animal from different groups was placed in Digital Photoactometer for 5 minutes and scores were recorded.

OTHER: No data

Sacrifice and pathology:

Sacrifice and pathology
GROSS PATHOLOGY: Yes
All surviving 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.

Organs weighed: Liver, Adrenals, Spleen, Heart, Kidney, Brain, Testes, Epididymides, Ovaries (incl. paired ovaries and uterus, including cervix), Thymus

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.

Other examinations:

No data

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.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

Soft stool, nasal discharge, red crust around nostrils, perineum soiled with fecal matter 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.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed in treated animals.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

In male animals, high-dose treatment resulted into significant elevation in body weights on the day 01, 08, 15 and 22. In female rats, significant increase in the body weight by high-dose group has been observed on day 01, 08, 15, 22, 28 and terminal day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No change in average food consumption was observed in 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 in average water consumption was observed in male and female rat of treated groups.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No abnormalities were observed in all treated dose groups rat.

Haematological findings:

no effects observed

Description (incidence and severity):

When treated with 1000 mg/kg/day, Lymphocyte and Basophils level was significantly increased.

When treated with 500 mg/kg/day, In male rats, MCV, Lymphocyte and Basophils level was significantly increased.

Platelet level was significantly decreased in 500 and 1000 mg/kg/day as comparison to control.

When treated with 250 mg/kg/day, platelet, monocytes, neutrophils and eosinophils were decreased significantly.

When treated with 1000 mg/kg/day, In female rats, Platelet counts were significantly decreased, MCHC and Basophils were significantly increased as compare to control.

When treated with 500 mg/kg/day, lymphocyte was significantly decreased and WBC, MCHC and Basophils significantly increased as compare to control

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

In male rats 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 treated 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 significantly 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):

effects observed, treatment-related

Description (incidence and severity):

There was statistically significant decrease observed in the motor activity scores for 250 mg/Kg bw dose group of male animals when compared with the control animals.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The absolute weight of brain was found to be increased significantly in all the dose treated groups. The low-dose treatment resulted in to the significant reduction in the absolute weight of adrenals along with the increased weight of spleen. However, the absolute weight of heart has also been increased significantly in 500 and 1000 mg/Kg bw dose treated animals, when compared to control group.

The relative organ weight of brain was significantly increased in 250 mg/kg bw dose group. However, it was decreased significantly in the animals treated with 1000 mg/Kg bw dose .The relative weights of liver, kidneys and adrenals were found to be decreased significantly by 250 mg/Kg bw treatment. The relative weight of kidneys was also decreased to significant levels in the 1000 mg/Kg bw-dose treated rats and significant reduction in the relative weight of epididymides was noticed in the 500 mg/Kg bw-dose treatment.

In female animals, the absolute weight of ovaries was found to be significantly reduced in all the treated groups in comparison to control. The absolute weight of spleen and heart has shown a significant increase by the 1000 mg/Kg bw-dose treatment. A significant elevation in the weight of uterus was noticed in the mid-dose treatment. The relative weight of liver was noticed to be decreased (p<0.05) in the 1000 mg/Kg bw-dose group. A significant reduction in the relative weight of ovaries was noticed in all the treated groups. The 1000 mg/Kg bw-dose also caused increase (p<0.05) in the relative weight of spleen. The relative weight of uterus was increased significantly (p<0.05) in the 250 mg/Kg bw dose treatment.

However, no microscopic abnormalities in both male and female animals were observed in respective organs in histological evaluations. Hence the changes in the organ weights have no toxicological relevance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

In terminally sacrificed rats, the incidence of necropsy findings included fat deposition around small, large intestine and abdominal cavity, tissue growth around heart, spotted lung and tumor like development (approximately 1 cm) in hind limb region (RHS). Above mentioned changes produced in organs might be due to adaptive metabolic and physiological changes, anoxic / hypoxic conditions during anesthesia and terminal sacrifice of the animals, which affect the normal structure of the organs and were considered to be dose independent. So, these findings were considered to be of no toxicological significance.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Focal fatty change in liver, reactive spleen and 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 dosed animals. However, these findings may not be linked with test item related effects as most of these have been observed in the control animals also. These types of findings may be considered to be within the range of normal background lesions which may be seen in rats of this strain and age of this study type and were considered incidental in nature with carbon dioxide inhalation and terminal changes at sacrifice, reflecting the usual individual variability.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

water consumption and compound intake

Remarks on result:

other: No effect observed

Target system / organ toxicity

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

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:

The No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg/bw/ day in male and female Sprague Dawley rats when exposed to test chemical Zinc Distearate (ZDS) (557-05-1) for 28 days by oral gavage.

Executive summary:

In a 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 were observed. Significant decreased were observed in water consumption and locomotor activity of female rat in 1000 mg/kg dose group. Bodyweight was increased significantly in male and female rats. Significant increase in MCV, Lymphocyte, Basophils level and significant decrease in WBC, platelet, monocytes, neutrophils and eosinophils were observed 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, the No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg/day when Sprague-Dawley rat exposed to test substance orally.