Reaction products of sodium glucoheptonate with zinc sulfate and sodium hydroxide

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1981

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

ICR mice and Wistar rats of both sexes were fed a diet containing ZnSO4 at 0, 300, 3, 000 and 30, 000 ppm for 13 weeks.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

zinc sulphate heptahydrate

Cas Number:

7446-20-0

IUPAC Name:

zinc sulphate heptahydrate

Test material form:

solid: crystalline

Specific details on test material used for the study:

heptahydrate zinc sulfate, ZnSO4. 7H2O

Test animals

Species:

other: rats and mice

Strain:

other: mice of the ICR strain and rats of the Wistar strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

Specific pathogen-free (SPF) mice of the ICR strain and rats of the Wistar strain were obtained at 4 weeks of age from Shizuoka Agriculture Cooperative Association for Laboratory Animals and used in the tests after an acclimatizing period of one week. The animals in groups of 4 of each sex were housed in stainless cages with a wire-meshed bottom. The animal rooms were sustained in a barrier system with a controlled temperature of 24+-1°C, humidity of 55+5% and light for 14 hrs a day. Food and water were supplied ad libitum. The basic diets used were pulverized chows, MF for mice and M for rats, produced by Oriental Yeast Co. ZnSO4 was mixed in the basic diet at the appropriate concentration levels and given to the animals.

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

ZnSO4 was mixed in the basic diet at the appropriate concentration levels and given to the animals.

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

When the diet was excessively discarded from the food jar by the animals, the discarded portion was dried and measured in order to obtain an accurate food intake.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

300 ppm

Doses / concentrations 2

Dose / conc.:

3 000 ppm

Doses / concentrations 3

Dose / conc.:

30 000 ppm

No. of animals per sex per dose:

12

Control animals:

yes, plain diet

Details on study design:

The animals were divided into four groups, each of which included 12 animals of each sex and fed one of the diets containing ZnSO4 at four different concentration levels, 0, 300, 3,000 and 30,000 ppm, for 13 weeks. The clinical signs of the animals were observed every day, they were weighed weekly and their diet and water intake measured twice a week. When the diet was excessively discarded from the food jar by the animals, the discarded portion was dried and measured in order to obtain an accurate food intake. Dead or moribund animals were necropsied as soon as possible and sampled for histopathological observations.

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: every day

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- 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: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: twice a week

HAEMATOLOGY: Yes
- Anaesthetic used for blood collection: Yes (light ether anesthesia)
- Animals fasted: Not specified
- How many animals: 12 per sex per dose
- erythrocyte count, hemoglobin and leukocyte count. Hematocrit was measured by the capillary tube method. A differential count of leukocyte (%) was performed from a blood smear from the tail vein.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Not specified
- How many animals: 12 per sex per dose
- total plasma protein, alkaline phosphatase, glucose, urea nitrogen, GOT, GPT, cholesterol and calcium

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
the following organs were weighed: brain, pituitary, thyroid, heart, thymus, liver, kidney, spleen, adrenals, gonads (testes or ovaries), and muscles (triceps surae).

HISTOPATHOLOGY: Yes
For histopathology additional organs and tissues were collected : submaxillary glands, lungs, mesentery lymph nodes, pancreas, stomach, small and large intestine, accessory genital organs, bone and bone marrow (sternum and femur), and lesions of gross abnormalities. Three or 4 µm paraffin sections from the specimens were stained with hematoxylineosin,periodic acid Schiff's reaction and azan for microscopic observations.

Statistics:

Student's t-test was used to estimate the statistical differences of the figures between controls and treated groups.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In mice, dead or moribund animals in the 30,000 ppm group showed depressed spontaneous motility and unkempt fur a little while before death or sacrifice.

Mortality:

mortality observed, treatment-related

Description (incidence):

In mice, four males and one female in the 30,000 ppm group were found dead or killed in extremis during the study. Mortality was 33. 3% in males and 8. 3% in females

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mice of both sexes in the 30, 000 ppm group showed a more prominently retarded growth resulting in smaller body size than did those of other groups. A significant but very slight depression of weight gain was seen in females of the 300 ppm group for a week after commencement, followed by a rapid recovery to the control level.
In rats of the 30, 000 ppm group a depressed weight gain and dwarfism similar to that observed in mice was seen in males. Weight gain of females in this group was slightly depressed during the study with significant differences to control animals in the 1st to 5th weeks of the study.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food intake of male and female mice in the 30,000 ppm group was depressed during the first week of the study in comparison to that of the controls but showed a tendency to recover afterwards. Average intake of these animals then remained at only a slightly lower level than that of the control group. In rats, the food intake of males decreased after the third week of the study in the 30,000 ppm group. A similar reduction was seen in females of this group during the 1st to 6th weeks but then disappeared. A slightly lower value of average food intake was disclosed in only males.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Food efficiencies of of male and female mice in the 30,000 ppm group were markedly lower than those of the control group during the first week of the study, corresponding to the decrease in food intake. The overall average food efficiency in the 30,000 ppm group was much lower than the control group. While there were some fluctuations in food efficiency of rats in each group, a slight reduction in overall average value was shown only in males of the 30, 000 ppm group.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Male and female mice in the 30, 000 ppm group showed moderately lower values in hematocrit and hemoglobin concentration than those of the control group; the leukocyte count in males also decreased moderately.
In rats, a moderate reduction in leukocyte count was shown in both sexes in the 30,000 ppm group; males also disclosed a slight decrease in hematocrit and hemoglobin concentration. There were no remarkable changes in animals in the less than 3,000 ppm group, but there was a slight increment of hemoglobin concentration in females in the 3,000 ppm group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Mice of both sexes in the 30,000 ppm group showed a slight to moderate decrease in total protein, glucose and cholesterol and a moderate to marked increase in alkaline phosphatase and urea nitrogen. Additional significant changes occurring in these animals were depression of GPT level and increase in calcium level in females, and an increase of GOT level in males.
Significant reductions or reductive tendencies were seen in rats in the following parameters: GOT and GPT in all male chemically fed groups, total protein, cholesterol and calcium levels in males in the 30,000 ppm group and calcium level in females in both the 3,000 and 30,000 ppm groups.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Male and female rats in the 30, 000 ppm group showed a symptom of discarding the diet from the food jar by picking it out with their fore-limbs. This symptom began a week after commencement of the experiment and persisted throughout the study.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Organ weight analysis of the mice revealed that coincidental increase in absolute and relative weight was found in the thyroids of males and the kidneys of females in the 30,000 ppm group.
In the organ weights of the rats, a slight or moderate decrease in absolute and relative weights was seen in the liver and kidney among males in the 30,000 ppm group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In mice, marked emaciation, ischemic discoloration of the kidney and thyroid, strophy of the pancreas, edematous thickening of the upper small intestine and slight splenomegaly were recorded in the 30, 000 ppm group at necropsy, in addition to several cases of forestomach ulcer.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Mice (30,000 ppm): impairment of the urinary tract and regressive changes in the exocrine gland of the pancreas. Morphological changes of erythrocyte, anisocytosis, polychromatophilia and poikilocytosis, were seen in 6 males and 4 females which were reported by necropsy or microscopic observation to have fore-stomach ulcers. There were lesions attributable to the treatment in the pancreas, upper intestine, stomach, spleen and kidney from mice in the 30,000 ppm group. The pancreatic acinar cells had swollen nuclei with an increased number of clarified nucleoii and whirl-like profiles in their cytoplasm which were more basophilically stained than the controls. Single cell necrosis of the acinar cells was also a common feature in these animals. Moreover, a decrease in the number of acinus and ductulelike metaplasia of acinar cells was demonstrated. There were mucosal catarrh in the upper intestine with proliferation of epithelial cells and edema at lamina propria, slight to moderate ulcerative lesions in the boundary of the fore-stomach and proliferation of erythropoietic immature cells in the splenic red pulp of these animals. Regressive changes of the renal cortex were observed in the females.
In rats, pancreatic lesions similar to those in mice were seen in the 30, 000 ppm group, as well as degeneration and necrosis of the acinar cells, clarification of centroacinar cells and interstitial fibrosis. No other lesions attributable to the treatment were found in rats.

Histopathological findings: neoplastic:

no effects observed

Effect levels

1

Dose descriptor:

NOEL

Remarks:

mice and rats

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

2

Dose descriptor:

NOEL

Remarks:

mice

Effect level:

458 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical biochemistry

gross pathology

haematology

organ weights and organ / body weight ratios

3

Dose descriptor:

NOEL

Remarks:

mice

Effect level:

479 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical biochemistry

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

4

Dose descriptor:

NOEL

Remarks:

rats

Effect level:

234 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical biochemistry

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

5

Dose descriptor:

NOEL

Remarks:

rats

Effect level:

243 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical biochemistry

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The maximum no-effect level of ZnSO4 was determined to be 3, 000 ppm, which is approximately equivalent to the following milligram doses: mice; male 458 mg/kg/day, female 479 mg/kg/day, rats; male 234 mg/kg/day, female 243 mg/kg/day.

Executive summary:

ICR mice and Wistar rats of both sexes were fed a diet containing ZnSO4 at 0, 300, 3,000 and 30,000 ppm for 13 weeks. Animals in the 30,000 ppm group showed retarded growth along with low food intake, abnormal values in a few hematological parameters and regressive changes of the pancreatic exocrine gland. In addition, mice had decreased water intake and significant deviations in biochemical parameters, toxic lesions appeared in the stomach, intestine and spleen of both sexes and in the kidney of females. Four males and one female mouse were found dead or moribund during the study. The maximum no-effect level of ZnSO4 was determined to be 3, 000 ppm, which is approximately equivalent to the following milligram doses: mice; male 458 mg/kg/day, female 479 mg/kg/day, rats; male 234 mg/kg/day, female 243 mg/kg/day.