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Administrative data

Description of key information

To address the endpoint repeated-dose toxicity, read-across on gluconates and derivatives and zinc compounds was performed within the frame of a weight-of-evidence approach.The underlying hypothesis for the read-across is that glucoheptonates and gluconates, structurally similar sugar-like carbohydrate metal-complexes, share the same metabolism pathways in mammals (they are oxidized by pentose phosphate pathway) and that their possible toxicity is a function of the metal cation rather than of the gluconate or glucoheptonate anion.

Therefore, data on gluconates, elemental zinc and zinc salts were taken into account to assess the repeated-dose toxicity of zinc glucoheptonate.

Data on Gluconates (SIDS, 2004)

rat

A 28-day study was conducted by feeding rats by gavage with sodium gluconate at doses of 0, 500, 1000, 2000 mg/kg bw in water at a volume of 1 mL/ 100g bw. No death or clinical signs of abnormality were observed in any of the groups. Histopathological examination showed a thickening of the limiting ridge of the stomach in 5 out of 12 males at 2000 mg/kg bw per day dose. No toxic changes associated with the test article were detected. As the limiting ridge is a tissue specific to rodents, this lesion is not toxicologically relevant for humans. Other lesions occurred incidentally and were not treatment -related. The NOAEL was estimated to be 1000 mg/kg bw/day for males and 2000 mg/kg bw/day for female.

Another 28-day toxicity study in rats fed with a diet containing up to 5% w/w sodium gluconate (max. 4100 mg/kg bw for males and 4400 mg/kg bw for females) was conducted using a control group receiving equivalent concentration of sodium in the form of NaCl in order to differentiate the potential effects of high doses of sodium intake. No deaths occurred during the study period. No revisions in the general condition, body weight, or food and water intake were observed in the animals over the study period. No changes were observed in the investigated ophthalmologic tests, urinalysis, hematology and blood chemistry over the study period. In addition, histopathological examination indicated no adverse effects as a result of the treatment regime. Statistically significant differences in some urinary parameters reported in animals receiving 2.5 or 5% sodium gluconate were comparable to those observed in the NaCl control group, and were interpreted as related to the high sodium concentration of the diet.

The authors concluded that the NOAEL was 5% (equal to 4100 mg/kg bw per day). However, The JECFA committee who evaluated this report has concluded that the study was not suitable for identifying a NOAEL because of the small group sizes and the positive findings in the qualitative analysis, even if they have acknowledged that the effects shown in the qualitative urine analyses were related to the high sodium intake. Nonetheless, this study demonstrates the lack of effects of the gluconate anion even in large doses as the urinary effects were attributed to the high sodium intake and was therefore considered as critical for this endpoint.

In a 6 months-study Glucono-delta-lactone (250, 500, 1000, 2000 and 4000 mg/kg bw) was orally administered to Sprague-Dawley rats. In all dose groups, thickening of the stratified squamous epithelium was detected at the anterior stomach, particularly the transitional area continuous with the pyloric stomach; the frequency and severity of this thickening increased with the dose. In high dose groups, submucosal inflammatory cell infiltration was also detected, but this change was not statistically significant. No deaths or other abnormalities were detected.

In Wistar rats fed for 24 months with a diet containing 2.5% and 10% of glucono-delta-lactone (the total intake of the drug : 1240-1350 mg/kg bw. in 2.5% GDL group and 4920-5760 mg/kg bw. in the 10 % GDL group), no changes were observed in the general condition throughout the period of testing, but weight gain tended to be slightly reduced 2-3 months after the initiation of the test feeding in 10% GDL group. There was no statistically significant difference in the number and time of deaths between the treated and control groups. Histopathological changes accompanying aging were observed in all groups including the controls, but no specific changes likely to be associated with the test substance were detected.

dog

Repeated toxicity studies were also performed on Beagle dogs with sodium gluconate administered orally for 4 weeks at 500, 1000, 2000 mg/kg bw. doses. None of the animals died during the period of treatment in any dose group and no significant toxicologically changes were detected in the body weight, food intake, water intake, urinalysis, haematological test, blood chemistry analysis, ophthalmologic test, electrocardiography, autopsy and organ weight or in histopathological examination. However, increased frequency of vomiting and loose or watery stools were observed in the 1000 and 2000 mg/kg bw. dose groups, as compared to controls.

On the basis of these results, the non-toxic dose was estimated to be 500 mg/kg bw / day. However, the toxicological effects observed (vomiting, passage of loose or watery stools) were considered extremely slight since other tests did not show the same changes.

These results are relevant for the target substance zinc glucoheptonate, because Gluconates are very similar to the glucoheptonate moiety of ZnGHA. Therefore, the fact that gluconates are not toxic underlines the assumption that the toxicity of ZnGHA is triggered by the Zn ion.

Data on elemental Zinc converted to zinc glucoheptonate

Data on elemental zinc allows estimating a corresponding effective dose for zinc glucoheptonate providing that no toxicity is attributed to the glucoheptonate ion, that the absorpton of zinc is 100% and all zinc became systemically available. 

The effective dose (rat, 4 weeks) for elemental zinc is reported to be 0.12 mg/ml drinking water which corresponds to an effective dose of 0.76 mg/ml drinking water for zinc glucoheptonate (Zaporowska and Wasilewski, 1992).

The effective dose (mouse, 14 months) for elemental zinc is reported to be 500 mg/l drinking water which corresponds to an effective dose of 3156.4 mg/l drinking water for zinc glucoheptonate (Aughey et al., 1977).

Data on Zinc salts converted to zinc glucoheptonate

Data on zinc acetate allow estimating a corresponding NOEL for zinc glucoheptonate providing that no toxicity is attributed to glucoheptonate or acetate ion, that the absorption of zinc from this zinc compounds is 100 % and all zinc became systemically available. 

The NOEL (mouse, 3 months) for zinc acetate is reported to be 160 mg/kg bw which corresponds to a NOEL of 360 mg/kg bw for zinc glucoheptonate (Llobet et al., 1988).

Data on ZnSO4 + 7 H2O allow estimating a corresponding effective dose for zinc glucoheptonate providing that no toxicity is attributed to glucoheptonate or sulfate ion, that the absorption of zinc from this zinc compounds is 100 % and all zinc became systemically available. 

The effective dose (mouse, 4 weeks) for ZnSO4 + 7 H2O is reported to be 2.6 g/kg ww which corresponds to an effective dose of 3.85 g/kg ww for zinc glucoheptonate (Schiffer et al., 1991).

A 13 weeks study with mice and rats with 300 to 30,000 ppm ZnSO4 was conducted (Maita et al. 1981). 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.

The maximum no-effect level of ZnGHA (mice and rats, 13 weeks) was determined to be 4355 ppm, which is approximately equivalent to the following milligram doses: mice: male 665 mg/kg/day, female 695 mg/kg/day, rats: male 339 mg/kg/day, female 353 mg/kg/day.

Conclusion

Based on the given information the NOAEL of 339 mg/kg bw/day, which is derived from data on ZnSO4 (Maita et al. 1981) is regarded as suitable hazard value for the assessment of the repeated-dose toxicity of the registered substance.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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
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
Specific details on test material used for the study:
heptahydrate zinc sulfate, ZnSO4. 7H2O
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.
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
Dose / conc.:
300 ppm
Dose / conc.:
3 000 ppm
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.
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.
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
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
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
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
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
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
Critical effects observed:
no
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.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable well documented peer-reviewed report.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Principles of method if other than guideline:
A 28-day study was conducted by feeding rats by gavage with sodium gluconate at doses of 0, 500, 1000, 2000 mg/kg bw in water at a volume of 1 ml/ 100g bw.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Route of administration:
oral: gavage
Details on route of administration:
feeding by gavage in water at a volume of 1 ml/ 100g bw.
Vehicle:
water
Details on oral exposure:
feeding by gavage in water at a volume of 1 ml/ 100g bw.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12/sex/group
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: not specified
- Rationale for animal assignment (if not random): not specified
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): not specified
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Not specified
- Time schedule:

BODY WEIGHT: Yes / No / Not specified
- Time schedule for examinations:

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 / No / Not specified

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Not specified
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Not specified
- Time schedule for collection of blood:
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No.?] were examined.

URINALYSIS: Not specified
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / Not specified
- Animals fasted: Yes / No / Not specified
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: Not specified
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: Not specified
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
not specified
Statistics:
no data
Clinical signs:
no effects observed
Description (incidence and severity):
No death or clinical signs of abnormality were observed in any of the groups.
Mortality:
no mortality observed
Description (incidence):
No death or clinical signs of abnormality were observed in any of the groups.
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Histopathological examination showed a thickening of the limiting ridge of the stomach in 5 out of 12 males at 2000 mg/kg bw per day dose. No toxic changes associated with the test article were detected. As the limiting ridge is a tissue
specific to rodents, this lesion is not toxicologically relevant for humans. Other lesions occurred incidentally and were not treatment -related.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
histopathology: non-neoplastic
mortality
Dose descriptor:
NOAEL
Effect level:
2 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
histopathology: neoplastic
mortality
Critical effects observed:
no

A 28-day study was conducted by feeding rats by gavage with sodium gluconate at doses of 0, 500, 1000, 2000 mg/kg bw in water at a volume of 1 ml/ 100g bw. No death or clinical signs of abnormality were observed in any of the groups. Histopathological examination showed a thickening of the limiting ridge of the stomach in 5 out of 12 males at 2000 mg/kg bw per day dose. No toxic changes associated with the test article were detected. As the limiting ridge is a tissue specific to rodents, this lesion is not toxicologically relevant for humans. Other lesions occurred incidentally and were not treatment -related.

The NOAEL was estimated to be 1000 mg/kg bw/day for males and 2000 mg/kg bw/day for female (Mochizuki, M, Bozo Research Center, 1995a).

Conclusions:
None of the repeated dose toxicity studies of any duration (4 weeks, 6 months, or 24 months) showed any significant toxicological effects of gluconates. Potential side effects were attributed to high doses of cation intake, evidenced by results from assays designed for the gluconate anion effect specifically. The NOAEL of sodium gluconate determined from the 28 days studies on rats was equal to 1000 mg/kg bw for males and 2000 mg/kg bw for females. On the basis of these data and considering that gluconates are used as food additives permitted in the EU following the Quantum Satis principle (no maximum level specified), further chronic toxicity tests are considered unnecessary.
Executive summary:

A 28-day study was conducted by feeding rats by gavage with sodium gluconate at doses of 0, 500, 1000, 2000 mg/kg bw in water at a volume of 1 mL/ 100g bw. No death or clinical signs of abnormality were observed in any of the groups. Histopathological examination showed a thickening of the limiting ridge of the stomach in 5 out of 12 males at 2000 mg/kg bw per day dose. No toxic changes associated with the test article were detected. As the limiting ridge is a tissue specific to rodents, this lesion is not toxicologically relevant for humans. Other lesions occurred incidentally and were not treatment -related. The NOAEL was estimated to be 1000 mg/kg bw/day for males and 2000 mg/kg bw/day for female (Mochizuki, M, Bozo Research Center, 1995a).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Concentration of glucose, GOT, GPT, ALP, urea and creatinine in plasma were measured according to clinical chemistry methods decribed by Domingo et al. (1985).
GLP compliance:
not specified
Limit test:
yes
Specific details on test material used for the study:
The test mateial is provided by Merck, Darmstadt, FRG.
Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Interfauna Iberica, Barcelona, Spain
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: not specified
- Weight at study initiation: 70 - 90g
- Fasting period before study: not specified
- Housing: individual metabolism cages
- Diet: ad libitum (Panlab diet, Barcelona, Spain)
- Water: ad libitum
- Acclimation period: not specified
Route of administration:
oral: drinking water
Vehicle:
water
Details on oral exposure:
Zinc acetate dihydrate (Merck, Darmstadt, FRG) was solved in the drinking water at levels of O, 160, 320 and 640 mg/kg body weight/day continously for three months. Solutions were prepared weekly to achieve a constant intake. Sugar was added to reduce the aversive effect of the zinc in the water. Similar quantities of sugar were also added to the drinking water of control animals in order to make comparable the results.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
3 months / exposure via drinking water
Frequency of treatment:
Water consumption was freely available.
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
160 mg/kg bw/day (nominal)
Dose / conc.:
320 mg/kg bw/day (nominal)
Dose / conc.:
640 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 female rats
Control animals:
yes
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes / No / Not specified
- Time schedule:
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes / No / Not specified
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: body weights of all rats were recorded prior to treatment and weekly throughout the study.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Food and water consumption were measured daily.

OPHTHALMOSCOPIC EXAMINATION: Yes / No / Not specified
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes / No / Not specified
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No.?] were examined.

The volume of urine and the weight of excreted feces were measured daily, at the end of the experiment, blood samples were collected from the tail vein. Hematocrit and hemoglobin determinations, as well as the concentration of glucose, GOT, GPT, ALP, urea and creatinine in plasma were also measured.
Sacrifice and pathology:
After three months of the treatment the animals were sacrificed and the weight of the brain, heart, lungs, spleen, liver and kidneys measured.

HISTOPATHOLOGY: Yes
Histological examinations (paraffin slices, hematoxilin-eosin) of spleen, liver, heart, lungs and kidneys were performed in three rats of each group.
Other examinations:
Zinc concentrations in brain, liver, kidneys, spleen, heart, lungs, bone (femur), abdominal muscle and blood were determined by atomic absorption spectrophotometry (Perkin-Elmer 4000).
Statistics:
Significance of the differences between the treatment groups were determined using the Student's t-test (p < 0.05).
Clinical signs:
not specified
Mortality:
mortality observed, treatment-related
Description (incidence):
Of the animals which received zinc actetate, 2 animals died during the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was no effect on weight gain caused by treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
The drinking water ingested and the volume of urine excreted were always significantly lower for the 640 mg / kg bw / day group.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
No signilicant differences were round in the mean values of hematocrit and hemoglobin. The concentrations of glucose, GOT, GPT and ALP in plasma were within the normal range for treated and untreated animals. However, the concentrations of urea and creatinine and plasma were significantly higher for the animais receiving 640 mg / kg bw / day zinc acetate than for the control rats.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The highest concentrations were found in bone, liver and kidneys as well as in blood. A significant relationship between dose received and tissue concentrations can be seen.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Rats receiving zinc acetate showed tendency towards apathy.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The most severe histological lesions were observed in kidneys. These lesions consisted in glomerular Bowman's capsule with flattened epithelial cells and proximal convoluted tubules with desquammtion of tubular epithelial cells and picnotic nucleus.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The most severe histological lesions were observed in kidneys. These lesions consisted in glomerular Bowman's capsule with flattened epithelial cells and proximal convoluted tubules with desquammtion of tubular epithelial cells and picnotic nucleus.
Histopathological findings: neoplastic:
not examined
Dose descriptor:
NOEL
Effect level:
160 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
water consumption and compound intake
Critical effects observed:
no
Conclusions:
Concentrations of urea and creatinine in plasma for the 640 mg / kg bw / day group were significantly increased compared with the control group. Thls fact suggests a remarkable effect on the renal function which has been corroborated by the histopathological study of the kidneys. The significant decreases in the volume of urine excreted for these animals could also suggest a disorder on the renal function. The results have demonstrated a toxicological no observable- effect level (NOEL) for zinc acetate of 160 mg /kg body weight / day in female rats.
Executive summary:

A total of forty female Sprague-Dawley rats were exposed to zinc acetate dihydrate in the drinking water at levels of 0, 160, 320 and 640 mg / kg body weight / day continously for three months. The animals were placed in individual metabolism cases. Body weights of all rats were recorded prior to treatment and weekly throughout the study. After three months, the rats were sacrificed.

There was no effect on weight gain caused by the treatment. No signilicant differences were found in the mean values of hematocrit and hemoglobin. The concentrations of glucose, GOT, GPT and ALP in plasma were within the normal range. The most severe histological lesions were observed in kidneys. The highest zinc concentrations were found in bone, liver and kidneys as well as in blood. A significant relationship between dose received and tissue concentrations can be seen. Concentrations of urea and creatinine in plasma for the 640 mg / kg bw / day group were significantly increased compared with the control group. This fact suggests a remarkable effect on the renal function which has been corroborated by the histopathological study of the kidneys. The significant decreases in the volume of urine excreted for these animals could also suggest a disorder on the renal function. This finding agrees with the significant lower ingestion of water in this group. The results have demonstrated a toxicological no observable - effect level (NOEL) for zinc acetate of 160 mg / kg body weight / day in female rats.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable well documented peer-reviewed report.
Principles of method if other than guideline:
Repeated toxicity studies were also performed on Beagle dogs with sodium gluconate administered orally for 4 weeks at 500, 1000, 2000 mg/kg bw. doses.
GLP compliance:
not specified
Limit test:
no
Species:
dog
Strain:
Beagle
Sex:
not specified
Route of administration:
oral: unspecified
Details on route of administration:
oral administration
Vehicle:
not specified
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
not specified (adminstered orally)
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
4/sex/group
Control animals:
yes
Details on study design:
- Dose selection rationale: not specified
- Rationale for animal assignment (if not random): not specified
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): not specified
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Not specified
- Time schedule:

BODY WEIGHT: Yes / No / Not specified
- Time schedule for examinations:

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 / No / Not specified

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Not specified
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Not specified
- Time schedule for collection of blood:
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No.?] were examined.

URINALYSIS: Not specified
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / Not specified
- Animals fasted: Yes / No / Not specified
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: Not specified
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: Not specified
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
not specified
Statistics:
no data
Clinical signs:
no effects observed
Description (incidence and severity):
Increased frequency of vomiting and loose or watery stools were observed in the 1000 and 2000 mg/kg bw. dose groups, as compared to controls.
Mortality:
no mortality observed
Description (incidence):
None of the animals died during the period of treatment in any dose group
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significantly toxicologically changes were detected in the body weight
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No significantly toxicologically changes were detected in the food intake
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No significantly toxicologically changes were detected in the water intake
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No significantly toxicologically changes were detected in the ophthalmologic test
Haematological findings:
no effects observed
Description (incidence and severity):
No significantly toxicologically changes were detected in the haematological test
Clinical biochemistry findings:
not specified
Description (incidence and severity):
No significantly toxicologically changes were detected in the blood chemistry analysis
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
No significantly toxicologically changes were detected in the urinalysis
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No significantly toxicologically changes were detected in the organ weight examination.
Gross pathological findings:
not specified
Description (incidence and severity):
No significantly toxicologically changes were detected in the autopsy examination.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No significantly toxicologically changes were detected in histopathological examination.
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Description (incidence and severity):
No significantly toxicologically changes were detected in the electrocardiography examination.
Details on results:
Repeated toxicity studies were also performed on Beagle dogs with sodium gluconate administered orally for 4 weeks at 500, 1000, 2000 mg/kg bw. doses. None of the animals died during the period of treatment in any dose group and no significantly toxicologically changes were detected in the body weight, food intake, water intake, urinalysis, haematological test, blood chemistry analysis, ophthalmologic test, electrocardiography, autopsy and organ weight or in histopathological examination. However, increased frequency of vomiting and loose or watery stools were observed in the 1000 and 2000 mg/kg bw. dose groups, as compared to controls.
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical signs
gross pathology
histopathology: non-neoplastic
mortality
ophthalmological examination
organ weights and organ / body weight ratios
urinalysis
water consumption and compound intake
Critical effects observed:
no

Repeated toxicity studies were also performed on Beagle dogs with sodium gluconate administered orally for 4 weeks at 500, 1000, 2000 mg/kg bw. doses. None of the animals died during the period of treatment in any dose group and no significantly toxicologically changes were detected in the body weight, food intake, water intake, urinalysis, haematological test, blood chemistry analysis, ophthalmologic test, electrocardiography, autopsy and organ weight or in histopathological examination. However, increased frequency of vomiting and loose or watery stools were observed in the 1000 and 2000 mg/kg bw. dose groups, as compared to controls.

On the basis of these results, the non-toxic dose was estimated to be 500 mg/kg bw / day. However, the toxicological effects observed (vomiting, passage of loose or watery stools) were considered extremely slight since other tests did not show the same changes (Okamoto, M. Bozo Research Center, 1995a).

Conclusions:
None of the repeated dose toxicity studies of any duration (4 weeks, 6 months, or 24 months) showed any significant toxicological effects of gluconates. Potential side effects were attributed to high doses of cation intake, evidenced by results from assays designed for the gluconate anion effect specifically. On the basis of these data and considering that gluconates are used as food additives permitted in the EU following the Quantum Satis principle (no maximum level specified), further chronic toxicity tests are considered unnecessary.
Executive summary:

Repeated toxicity studies were also performed on Beagle dogs with sodium gluconate administered orally for 4 weeks at 500, 1000, 2000 mg/kg bw. doses. None of the animals died during the period of treatment in any dose group and no significantly toxicologically changes were detected in the body weight, food intake, water intake, urinalysis, haematological test, blood chemistry analysis, ophthalmologic test, electrocardiography, autopsy and organ weight or in histopathological examination. However, increased frequency of vomiting and loose or watery stools were observed in the 1000 and 2000 mg/kg bw. dose groups, as compared to controls.

On the basis of these results, the non-toxic dose was estimated to be 500 mg/kg bw / day. However, the toxicological effects observed (vomiting, passage of loose or watery stools) were considered extremely slight since other tests did not show the same changes (Okamoto, M. Bozo Research Center, 1995a).

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable well documented peer-reviewed report.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Principles of method if other than guideline:
A 28-day oral feeding study was conducted rats with sodium gluconate at doses of 0, 1000, 2000 and 4100 mg/kg bw.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
oral: feed
Details on route of administration:
oral feeding
Vehicle:
not specified
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Dose / conc.:
4 100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10/sex/group
Control animals:
yes
Details on study design:
- Dose selection rationale: not specified
- Rationale for animal assignment (if not random): not specified
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): not specified
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Not specified
- Time schedule:

BODY WEIGHT: Yes / No / Not specified
- Time schedule for examinations:

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 / No / Not specified

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Not specified
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Not specified
- Time schedule for collection of blood:
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No.?] were examined.

URINALYSIS: Not specified
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / Not specified
- Animals fasted: Yes / No / Not specified
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: Not specified
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: Not specified
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
not specified
Statistics:
no data
Clinical signs:
no effects observed
Description (incidence and severity):
No revisions in the general condition were observed in the animals over the study period.
Mortality:
no mortality observed
Description (incidence):
No deaths occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No revisions in the body weight were observed in the animals over the study period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No revisions in the food intake were observed in the animals over the study period.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No revisions in the water intake were observed in the animals over the study period.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No changes were observed in the investigated ophthalmologic tests
Haematological findings:
no effects observed
Description (incidence and severity):
No changes were observed in the investigated hematology
Clinical biochemistry findings:
not specified
Description (incidence and severity):
No changes were observed in the investigated blood chemistry
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant differences in some urinary parameters reported in animals receiving 2.5 or 5% sodium gluconate were comparable to those observed in the NaCl control group, and were interpreted as related to the high sodium concentration of the diet.
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histopathological examination indicated no adverse effects as a result of the treatment regime
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
No deaths occurred during the study period. No revisions in the general condition, body weight, or food and water intake were observed in the animals over the study period. No changes were observed in the investigated ophthalmologic tests, urinalysis, hematology and blood chemistry over the study period. In addition, histopathological examination indicated no adverse effects as a result of the treatment regime. Statistically significant differences in some urinary parameters reported in animals receiving 2.5 or 5% sodium gluconate were comparable to those observed in the NaCl control group, and were interpreted as related to the high sodium concentration of the diet.
Dose descriptor:
NOAEL
Effect level:
4 100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
clinical signs
gross pathology
histopathology: non-neoplastic
mortality
ophthalmological examination
organ weights and organ / body weight ratios
urinalysis
water consumption and compound intake
Remarks on result:
other: 5% w/w sodium gluconate (max. 4100 mg/kg bw for males and 4400 mg/kg bw for females)
Dose descriptor:
NOAEL
Effect level:
4 400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
clinical signs
gross pathology
histopathology: non-neoplastic
mortality
ophthalmological examination
organ weights and organ / body weight ratios
urinalysis
water consumption and compound intake
Remarks on result:
other: 5% w/w sodium gluconate (max. 4100 mg/kg bw for males and 4400 mg/kg bw for females)
Critical effects observed:
no

A 28-day toxicity study in rats fed with a diet containing up to 5% w/w sodium gluconate (max. 4100 mg/kg bw for males and 4400 mg/kg bw for females) was conducted using a control group receiving equivalent concentration of sodium in the form of NaCl in order to differentiate the potential effects of high doses of sodium intake. No deaths occurred during the study period. No revisions in the general condition, body weight, or food and water intake were observed in the animals over the study period. No changes were observed in the investigated ophthalmologic tests, urinalysis, hematology and blood chemistry over the study period. In addition, histopathological examination indicated no adverse effects as a result of the treatment regime. Statistically significant differences in some urinary parameters reported in animals receiving 2.5 or 5% sodium gluconate were comparable to those observed in the NaCl control group, and were interpreted as related to the high sodium concentration of the diet.

The authors concluded that the NOAEL was 5% (equal to 4100 mg/kg bw per day). However, The JECFA committee who evaluated this report has concluded that the study was not suitable for identifying a NOAEL because of the small group sizes and the positive findings in the qualitative analysis, even if they have acknowledged that the effects shown in the qualitative urine analyses were related to the high sodium intake (Mochizuki, M. Bozo Research Center, 1997). Nonetheless, this study demonstrates the lack of effects of the gluconate anion even in large doses as the urinary effects were attributed to the high sodium intake and was therefore considered as critical for this endpoint.

Conclusions:
None of the repeated dose toxicity studies of any duration (4 weeks, 6 months, or 24 months) showed any significant toxicological effects of gluconates. Potential side effects were attributed to high doses of cation intake, evidenced by results from assays designed for the gluconate anion effect specifically. On the basis of these data and considering that gluconates are used as food additives permitted in the EU following the Quantum Satis principle (no maximum level specified), further chronic toxicity tests are considered unnecessary.
Executive summary:

Another 28-day toxicity study in rats fed with a diet containing up to 5% w/w sodium gluconate (max. 4100 mg/kg bw for males and 4400 mg/kg bw for females) was conducted using a control group receiving equivalent concentration of sodium in the form of NaCl in order to differentiate the potential effects of high doses of sodium intake. No deaths occurred during the study period. No revisions in the general condition, body weight, or food and water intake were observed in the animals over the study period. No changes were observed in the investigated ophthalmologic tests, urinalysis, hematology and blood chemistry over the study period. In addition, histopathological examination indicated no adverse effects as a result of the treatment regime. Statistically significant differences in some urinary parameters reported in animals receiving 2.5 or 5% sodium gluconate were comparable to those observed in the NaCl control group, and were interpreted as related to the high sodium concentration of the diet.

The authors concluded that the NOAEL was 5% (equal to 4100 mg/kg bw per day). However, The JECFA committee who evaluated this report has concluded that the study was not suitable for identifying a NOAEL because of the small group sizes and the positive findings in the qualitative analysis, even if they have acknowledged that the effects shown in the qualitative urine analyses were related to the high sodium intake (Mochizuki, M. Bozo Research Center, 1997). Nonetheless, this study demonstrates the lack of effects of the gluconate anion even in large doses as the urinary effects were attributed to the high sodium intake and was therefore considered as critical for this endpoint.

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1977
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
0.5 g/l zinc in the form of zinc sulphate was provided in distilled water for drinking ad libitum to mice. Untreated distilled water was available for the control animals. The mice were maintained for up to 14 months before killing. Control and test animals were removed from the colonies in groups of five, usually at monthly intervals, for the estimations of several parameters.
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
C3H
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Glasgow Institute of Radiotherapeutics and Oncology
- Age at study initiation: 6 weeks to 2 months
- Housing: not more than 15 animals were held in a cage at any time, sexes were kept separate.
- Diet: ad libitum, Oxoid breeding diet
- Water: ad libitum
- 150 mice in total
Route of administration:
oral: drinking water
Details on route of administration:
0.5 g zinc /l was added to distilled water for drinking ad libitum in the form of zinc sulphate.
Vehicle:
water
Remarks:
distilled water
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
An estimate of the amount of zinc swallowed by animals was difficult due to spillage during drinking and uncertain intestinal absorption.
Duration of treatment / exposure:
The mice were kept for 14 months before killing. However, usually at monthly intervals, groups of five animals of control and test animals were removed for examination.
Dose / conc.:
500 mg/L drinking water
No. of animals per sex per dose:
not specified
Control animals:
yes
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Not specified

DETAILED CLINICAL OBSERVATIONS: Yes / No / Not specified
- Time schedule:

BODY WEIGHT: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): How much the animals drank could only be estimated because of spillage during drinking.

Examination at monthly intervals of the plasma zinc, plasma glucose and insulin, tissue zinc of skin, liver and spleen, histological examination of small blocks of adrenal, pancreas and hypophysis cerebri were conducted on 5 animals per group. Also blocks of adrenal were analysed histochemically and sections from adrenal, pancreas and adenohypophysis were examined with a electron microscope.
Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
High level of appetite throughout the experiment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Description (incidence and severity):
How much the animals drank could only be estimated because of spillage during drinking.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The plasma insulin and glucose in mice supplemented by zinc were not signiicantly different from control values.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The prolonged administration of subtoxic quantities of zinc to mice affected the adrenal cortex, the pancreativ islets and the cells of the anterior lobe of the pituitary gland. All cell types in the pituitary specimen showed increase in size with evidence suggesting increase in both synthetic and secretory activities. The pancreatic islets and the zona fasciculata showed hypertrophy.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
The change in plasma zinc of zinc supplemented mice rised to 31 µM within 3 days of dosage. The total observation period of plasma zinc of zinc supplemented mice was 34 days, thereby, after the initial peak, the plasma zinc concentration dropped significantly. The mean plasma zinc level of the control mice was 15 +/- 1.35 µM thoughout the whole experiment.
The examination of tissue zinc concentration of liver, spleen and skin of zinc supplemented animals did not differ significantly from control animals over a period of 6 months.
Dose descriptor:
conc. level: leading to histopathological, non-neoplastic findings.
Effect level:
500 mg/L drinking water
Based on:
element
Sex:
male/female
Basis for effect level:
clinical biochemistry
histopathology: non-neoplastic
Critical effects observed:
no
Conclusions:
The results of this study indicate that the tested concentration of 500 mg zinc/ l drinking water, administered over a period of 14 months to mice lead to histopathological, non-neoplastic findings.
Executive summary:

In total, the effect of zinc (as ZnSO4) administered to mice by dosage of the distilled drinking water (500 mg Zinc/l) was tested. The 150 test animals were either assigned tothe group receiving the zinc supplementation or to the control group. The mice were kept for 14 months before killing. However, usually at monthly intervals, groups of five animals of control and test animals were removed for examination. An estimate of the amount of zinc swallowed by animals was difficult due to spillage during drinking and uncertain intestinal absorption.

The change in plasma zinc of zinc supplemented mice rised to 31 µM within 3 days of dosage. The total observation period of plasma zinc of zinc supplemented mice was 34 days, thereby, after the initial peak, the plasma zinc concentration dropped significantly. The mean plasma zinc level of the control mice was 15 +/- 1.35 µM thoughout the whole experiment. The examination of tissue zinc concentration of liver, spleen and skin of zinc supplemented animals did not differ significantly from control animals over a period of 6 months. The plasma insulin and glucose in mice supplemented by zinc were not signiicantly different from control values. The prolonged administration of subtoxic quantities of zinc to mice affected the adrenal cortex, the pancreativ islets and the cells of the anterior lobe of the pituitary gland. All cell types in the pituitary specimen showed increase in size with evidence suggesting increase in both synthetic and secretory activities. The pancreatic islets and the zona fasciculata showed hypertrophy.

The results of this study indicate that the tested concentration of 500 mg zinc/ l drinking water, administered over a period of 14 months to mice lead to histopathological, non-neoplastic findings.

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable well documented peer-reviewed report.
Principles of method if other than guideline:
Wistar rats were fed for 24 months with a diet containing 2.5% and 10% of glucono-delta-lactone (the total intake of the drug : 1240-1350 mg/kgbw. in 2.5% GDL group and 4920-5760 mg/kgbw. in the 10 % GDL group).
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Route of administration:
oral: feed
Vehicle:
not specified
Details on oral exposure:
a diet containing 2.5% and 10% of glucono-delta-lactone (the total intake of the drug : 1240-1350 mg/kgbw. in 2.5% GDL group and 4920-5760 mg/kgbw. in the 10 % GDL group)
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
24 months
Frequency of treatment:
continously
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
1 240 mg/kg bw/day (nominal)
Remarks:
1240-1350 mg/kgbw. in 2.5% GDL group
Dose / conc.:
4 920 mg/kg bw/day (nominal)
Remarks:
4920-5760 mg/kgbw. in the 10 % GDL group
No. of animals per sex per dose:
not specified
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: not specified
- Rationale for animal assignment (if not random): not specified
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): not specified
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Not specified
- Time schedule:

BODY WEIGHT: Yes / No / Not specified
- Time schedule for examinations:

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 / No / Not specified

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Not specified
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Not specified
- Time schedule for collection of blood:
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No.?] were examined.

URINALYSIS: Not specified
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / Not specified
- Animals fasted: Yes / No / Not specified
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: Not specified
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: Not specified
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
not specified
Statistics:
no data
Clinical signs:
no effects observed
Description (incidence and severity):
no changes were observed in the general condition throughout the period of testing
Mortality:
no mortality observed
Description (incidence):
There was no statistically significant difference in the number and time of deaths between the treated and control groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
weight gain tended to be slightly reduced 2-3 months after the initiation of the test feeding in 10% GDL group
Food consumption and compound intake (if feeding study):
not examined
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
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Histopathological changes accompanying aging were observed in all groups including the controls, but no specific changes likely to be associated with the test substance were detected (Fukuhara K, 1978a).
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
In Wistar rats fed for 24 months with a diet containing 2.5% and 10% of glucono-delta-lactone (the total intake of the drug: 1240-1350 mg/kgbw. in 2.5% GDL group and 4920-5760 mg/kgbw. in the 10 % GDL group), no changes were observed in the general condition throughout the period of testing, but weight gain tended to be slightly reduced 2-3 months after the initiation of the test feeding in 10% GDL group. There was no statistically significant difference in the number and time of deaths between the treated and control groups. Histopathological changes accompanying aging were observed in all groups including the controls, but no specific changes likely to be associated with the test substance were detected (Fukuhara K, 1978a).
Dose descriptor:
NOAEL
Remarks on result:
not determinable
Critical effects observed:
no

In Wistar rats fed for 24 months with a diet containing 2.5% and 10% of glucono-delta-lactone (the total intake of the drug : 1240-1350 mg/kgbw. in 2.5% GDL group and 4920-5760 mg/kgbw. in the 10 % GDL group), no changes were observed in the general condition throughout the period of testing, but weight gain tended to be slightly reduced 2-3 months after the initiation of the test feeding in 10% GDL group. There was no statistically significant difference in the number and time of deaths between the treated and control groups. Histopathological changes accompanying aging were observed in all groups including the controls, but no specific changes likely to be associated with the test substance were detected (Fukuhara K, 1978a).

Conclusions:
None of the repeated dose toxicity studies of any duration (4 weeks, 6 months, or 24 months) showed any significant toxicological effects of gluconates. Potential side effects were attributed to high doses of cation intake, evidenced by results from assays designed for the gluconate anion effect specifically. The NOAEL of sodium gluconate determined from the 28 days studies on rats was equal to 1000 mg/kg bw for males and 2000 mg/kg bw for females. On the basis of these data and considering that gluconates are used as food additives permitted in the EU following the Quantum Satis principle (no maximum level specified), further chronic toxicity tests are considered unnecessary.
Executive summary:

In Wistar rats fed for 24 months with a diet containing 2.5% and 10% of glucono-delta-lactone (the total intake of the drug : 1240-1350 mg/kgbw. in 2.5% GDL group and 4920-5760 mg/kgbw. in the 10 % GDL group), no changes were observed in the general condition throughout the period of testing, but weight gain tended to be slightly reduced 2-3 months after the initiation of the test feeding in 10% GDL group. There was no statistically significant difference in the number and time of deaths between the treated and control groups. Histopathological changes accompanying aging were observed in all groups including the controls, but no specific changes likely to be associated with the test substance were detected (Fukuhara K, 1978a).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable well documented peer-reviewed report.
Principles of method if other than guideline:
Glucono-delta-lactone (250, 500, 1000, 2000 and 4000 mg/kgbw. for 6 months) was orally administered to Sprague-Dawley rats.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
oral: unspecified
Vehicle:
not specified
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
6 months
Frequency of treatment:
not specified
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Dose / conc.:
4 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
not specified
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: not specified
- Rationale for animal assignment (if not random): not specified
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): not specified
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Not specified
- Time schedule:

BODY WEIGHT: Yes / No / Not specified
- Time schedule for examinations:

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 / No / Not specified

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Not specified
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Not specified
- Time schedule for collection of blood:
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No.?] were examined.

URINALYSIS: Not specified
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / Not specified
- Animals fasted: Yes / No / Not specified
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: Not specified
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: Not specified
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
not specified
Statistics:
no data
Clinical signs:
no effects observed
Description (incidence and severity):
No deaths or other abnormalities were detected
Mortality:
no mortality observed
Description (incidence):
No deaths or other abnormalities were detected
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not examined
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
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
In all dose groups, thickening of the stratified squamous epithelium was detected at the anterior stomach, particularly the transitional area continuous with the pyloric stomach; the frequency and severity of this thickening increased with the dose. In high dose groups, submucosal inflammatory cell infiltration was also detected, but this change was not statistically significant.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
In all dose groups, thickening of the stratified squamous epithelium was detected at the anterior stomach, particularly the transitional area continuous with the pyloric stomach; the frequency and severity of this thickening increased with the dose. In high dose groups, submucosal inflammatory cell infiltration was also detected, but this change was not statistically significant. No deaths or other abnormalities were detected (Fukuhara K, 1978).
Dose descriptor:
NOAEL
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no

Glucono-delta-lactone (250, 500, 1000, 2000 and 4000 mg/kgbw. for 6 months) was orally administered to Sprague-Dawley rats. In all dose groups, thickening of the stratified squamous epithelium was detected at the anterior stomach, particularly the transitional area continuous with the pyloric stomach; the frequency and severity of this thickening increased with the dose. In high dose groups, submucosal inflammatory cell infiltration was also detected, but this change was not statistically significant. No deaths or other abnormalities were detected (Fukuhara K, 1978). In Wistar rats fed for 24 months with a diet containing 2.5% and 10% of glucono-delta-lactone (the total intake of the drug : 1240-1350 mg/kgbw. in 2.5% GDL group and 4920-5760 mg/kgbw. in the 10 % GDL group), no changes were observed in the general condition throughout the period of testing, but weight gain tended to be slightly reduced 2-3 months after the initiation of the test feeding in 10% GDL group. There was no statistically significant difference in the number and time of deaths between the treated and control groups. Histopathological changes accompanying aging were observed in all groups including the controls, but no specific changes likely to be associated with the test substance were detected (Fukuhara K, 1978a).

Conclusions:
None of the repeated dose toxicity studies of any duration (4 weeks, 6 months, or 24 months) showed any significant toxicological effects of gluconates. Potential side effects were attributed to high doses of cation intake, evidenced by results from assays designed for the gluconate anion effect specifically. The NOAEL of sodium gluconate determined from the 28 days studies on rats was equal to 1000 mg/kg bw for males and 2000 mg/kg bw for females. On the basis of these data and considering that gluconates are used as food additives permitted in the EU following the Quantum Satis principle (no maximum level specified), further chronic toxicity tests are considered unnecessary.
Executive summary:

Glucono-delta-lactone (250, 500, 1000, 2000 and 4000 mg/kgbw. for 6 months) was orally administered to Sprague-Dawley rats. In all dose groups, thickening of the stratified squamous epithelium was detected at the anterior stomach, particularly the transitional area continuous with the pyloric stomach; the frequency and severity of this thickening increased with the dose. In high dose groups, submucosal inflammatory cell infiltration was also detected, but this change was not statistically significant. No deaths or other abnormalities were detected (Fukuhara K, 1978). In Wistar rats fed for 24 months with a diet containing 2.5% and 10% of glucono-delta-lactone (the total intake of the drug : 1240-1350 mg/kgbw. in 2.5% GDL group and 4920-5760 mg/kgbw. in the 10 % GDL group), no changes were observed in the general condition throughout the period of testing, but weight gain tended to be slightly reduced 2-3 months after the initiation of the test feeding in 10% GDL group. There was no statistically significant difference in the number and time of deaths between the treated and control groups. Histopathological changes accompanying aging were observed in all groups including the controls, but no specific changes likely to be associated with the test substance were detected (Fukuhara K, 1978a).

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
supplier: Fluka, Switzerland
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 2 month
- Weight at study initiation: females approximately 150g; males approximately 190g
- Housing: seperately in cages
- Diet: ad libitum, standard granulated rodent laboratory chow -LSM (CLPP, Motycz near Lublin)
- Water: ad libitum, test material administered with drinking water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 20 °C
- Humidity (%): 60 +/- 10%
- Photoperiod (hrs dark / hrs light): natural day-night light cycles

Route of administration:
oral: drinking water
Vehicle:
water
Details on oral exposure:
gavage of either 0.3 mg Vanadium/cm³ or 0.12 mg Zn/cm³ with drinking water or simultaneous gavage of vanadium and zinc with drinking water at the mentioned concentrations.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
4 weeks
Dose / conc.:
0.12 other: mg Zn / ml
Dose / conc.:
0.12 other: mg Zn / ml
Remarks:
addition of 0.3 mg V/ ml
Dose / conc.:
0.3 other: mg V / ml
No. of animals per sex per dose:
The exact number is not specified. However, it is mentioned that the 54 male and 64 female rats are, within each sex, randomly divided into four groups.
Control animals:
yes
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: not specified

BODY WEIGHT: Yes

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 fluid intake per day per kg body weight.

FOOD EFFICIENCY:
- Body weight gain recorded

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily recording of fluid intake

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4 weeks treatment
- Anaesthetic used for blood collection: Yes (ether anaesthesia)
- Animals fasted: Not specified
- How many animals: 106
Statistics:
The results were subject to statistical analysis (t-Test with P < 0.05).
Clinical signs:
not specified
Mortality:
mortality observed, treatment-related
Description (incidence):
In total, 5 male and 7 female rats died. Of the female rats, 3 were treated with vanadium spiked drinking water, 4 were treated with drinking water which was enriched with vanadium and zinc. Of the male rats, 2 were treated with vanadium spiked drinking water, 3 were treated with drinking water which was enriched with vanadium and zinc.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In general, a mean gain in body weight was observed for all groups. For some individuals a decrese in body weight was observed. The increment of body weight of animals given the aquaeous solution of zinc chloride was similar to that of the controls. Both groups which received aqueous solution with vanadium showed a statistically significant decrease in body weight increment compared to the control group.
The food consumption of the groups treated with either zinc, vanadium or the combination of zinc and vanadium in drinking water consumed less food than the control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The food consumption of the groups treated with either zinc, vanadium or the combination of zinc and vanadium in drinking water consumed less food than the control group.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
The fluid intake of the groups treated with either zinc, vanadium or the combination of zinc and vanadium in drinking water drank less water than the control group.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Animals receiving aqueous zinc showed a decrease in erythrocyte count and haemoglobin level in peripheral blood similar to the animals which received vanadium with their drinking water. The simultaneous gavage of zinc and vanadium did not noticeably deepen these changes. Also, an increase in lymphocyte count is observed for the zinc-receiving animals as well as the one which received zinc and vanadium simultaneously. The haematocrit index fell slightly after the 4 week treatment, but the percentage of reticulocytes and polychromatophilic erythrocytes in peripheral blood increased significantly compared to the control animals. In rats receiving inc or zinc and vanadium, the leukocyte count was increased compared to that of the control group. The analysis of bone marrow did not show major changes in comparison to the control group.
Clinical biochemistry findings:
not specified
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
For animals receiving zinc or the combination of zinc and vanadium diarrhoea was frequently observed.
Dose descriptor:
conc. level: leading to histopathological findings
Effect level:
120 mg/L drinking water
Based on:
element
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
haematology
mortality
water consumption and compound intake
other: diarrhoea
Critical effects observed:
no
Conclusions:
The tested concentration of 0.12 mg Zn / ml drinking water lead to histopathological findings.
Executive summary:

Within this study 2-month old rats (54 males, 64 females) were treated with either 0.3 mg Vanadium/cm³ or 0.12 mg Zn/cm³ with drinking water or simultaneous gavage of vanadium and zinc with drinking water at the mentioned concentrations over a time period of 4 weeks. The study was conducted in accordance with OECD Guideline 407 with minor adjustments. Of the female rats, 3 were treated with vanadium spiked drinking water, 4 were treated with drinking water which was enriched with vanadium and zinc, died. Of the male rats, 2 were treated with vanadium spiked drinking water, 3 were treated with drinking water which was enriched with vanadium and zinc, died. Because of these findings, it is assumed that the mortality is attributed to vanadium.

In general, a mean gain in body weight was observed for all groups.For some individuals a decrese in body weight was observed. The increment of body weight of animals given the aquaeous solution of zinc chloride was similar to that of the controls. Both groups which received aqueous solution with vanadium showed a statistically significant decrease in body weight increment compared to the control group. The food consumption of the groups treated with either zinc, vanadium or the combination of zinc and vanadium in drinking water consumed less food than the control group. Animals receiving aqueous zinc showed a decrease in erythrocyte count and haemoglobin level in peripheral blood similar to the animals which received vanadium with their drinking water. The simultaneous gavage of zinc and vanadium did not noticeably deepen these changes. Also, an increase in lymphocyte count is observed for the zinc-receiving animals as well as the one which received zinc and vanadium simultaneously. The haematocrit index fell slightly after the 4 week treatment, but the percentage of reticulocytes and polychromatophilic erythrocytes in peripheral blood increased significantly compared to the control animals. In rats receiving zinc or zinc and vanadium, the leukocyte count was increased compared to that of the control group. The analysis of bone marrow did not show major changes in comparison to the control group. Because haematological effects were observed for the treated animals, the tested concentration of 0.12 mg Zn / ml drinking water lead to histopathological findings.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
ZnSO4 * 7 H2O was used.
Species:
mouse
Strain:
other: SJL
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Jackson Laboratories (Bar Harbor, MI, U.S.A.)
- Age at study initiation: 4 weeks
- Housing: The animals were housed five per cage. The cages were
plastic tubs with wood chip bedding.
- Diet: not specified
- Water: doubly distilled water ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): not specified
- Humidity (%): not specified
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): Natural day-night cycles were simulated by vivarium lighting
Route of administration:
oral: feed
Details on oral exposure:
"The control diet was a high protein casein diet with vitamins, mineral supplementation, 0.03 g/kg zinc sulfate, and less than 0.01 g/kg nickel sulfate. A high zinc diet was formulated using the control diet plus 0.6 g/kg zinc sulfate. The zinc supplemented diet provides 2.6 g/kg wet weight (ZnSO4 • 7 H20) of zinc sulfate, and the nickel supplemented diet provides 2.7 g/kg wet weight (NiSO4 • 6 H20) of nickel sulfate. A fourth diet was also prepared consisting of the control diet supplemented both with NiSO4 and ZnSO4 in the above amounts. The diets were made to specification in pellet form by the Teklad Corporation, Madison, WI, U.S.A."
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
The animals were solely feed their respective diet.
Dose / conc.:
0.03 other: g/kg diet
Remarks:
and less than 0.01 g NiSO4 * 6 H2O/kg diet
Dose / conc.:
2.6 other: g/ kg wet weight
Remarks:
and less than 0.01 g NiSO4 * 6 H2O/kg diet
Dose / conc.:
2.6 other: g/kg wet weight
Remarks:
and 2.7 g NiSO4 * 6 H2O/kg wet weight
No. of animals per sex per dose:
Fifty animals were entered into the four dietary groups: 15 controls (low zinc and low nickel concentration), 15 high nickel diet, ten high zinc diet, and ten combined nickel/zinc diet.
Control animals:
yes
Observations and examinations performed and frequency:
Handling of SJL mice in this experiment was the minimal amount involved in daily checking.
Sacrifice and pathology:
HISTOPATHOLOGY: Yes
The lung, brain, liver and kidney were studied. Histopathologic assessment was performed in a blinded and standardized way upon 18 animals selected randomly from the four groups.
Other examinations:
All mice were immunized intraperitoneally with keyhole limpet hemocyanin (KLH) at 8 weeks. Eight days later, antibody responses to KLH were measured.
Statistics:
ANOVA and Student's t-test were performed.
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Within the groups that were supplemented with NiSO4, unexpected mortality occured. Thereby, 3 out of the 15 animals that were feed with the supplement of 2.7 g NiSO4 * 6 H2O and 2 out of 15 animals that were treated with high zinc and high nickel died.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
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:
effects observed, treatment-related
Description (incidence and severity):
No significant accumulation of elemental zinc occurred in brain and lung compared with control and nickel fed animals. Significant liver and kidney accumulations are observed in both zinc fed groups, compared with comparison groups (p = 0.02 or better, Mann-Whitney test).
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Within the control group in 3 of 5 mice examined, the lungs showed mild to moderate chronic alveolitis but no significant lesions were noted in other tissues.
In total 3 of 4 mice examined of the high nickel supplement group, pulmonary lesions were noted but no significant lesions were noted in other tissues.
The high Zn supplement group showed within 5 of 5 mice examined pulmonary lesions (three mice with mild to moderate interstitial pneumonitis, two with chronic alveolitis) and in 2 of 5 mice inflammatory cell infiltrates of the dermis were evident.
Regarding the mice treated with the high Zn, high Ni supplement diet, 2 of 4 examined mice showed alveolitis in the lungs and mild sinus histiocytosis was noted in a lymph node of one mouse.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
In the zinc supplemented animals alterations of antibody response or B cell reactivity were not found compared with controls. However, that T cell mitogenic responses of cultured splenocytes from zinc supplemented animals were enhanced compared with controls.
Dose descriptor:
conc. level: leading to histopathological findings
Effect level:
2.6 other: g/kg wet weight
Based on:
test mat.
Remarks:
ZnSO4 * 7 H2O
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
no
Conclusions:
The tested ZnSO4 * 7 H2O concentration of 2.6 g/ kg wet weight induced histopathological findings in mice.
Executive summary:

"SJL-strain female mice were fed with supplemental nickel and zinc sulfate from 4-8 weeks of age, and they were immunized intraperitoneally with keyhole limpet hemocyanin (KLH) at 8 weeks. Eight days later, antibody responses to KLH were measured. Zinc supplemented diets did not seem to affect antibody responsiveness and proliferation. The proliferative responses of B cells to the mitogen lipopolysaccharide (LPS) were not affected in the zinc fed animals. T cell mitogenic responses to concanavalin A were enhanced in zinc fed animals." The tested ZnSO4 * 7 H2O concentration of 2.6 g/ kg wet weight induced histopathological findings in mice.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
339 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The quality of the whole database is considered sufficient for estimation of repeated doese toxicity potential, because of the multipicity of available data for the different read-across substances

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

On the basis of the available data (NOAEL exceeds the cut-off value of 300 mg/kg bw/day), the registered substance does not require classification for lethal effects following a repeated exposure according to Regulation (EC) No 1272/2008.