Zinc sulphate

Repeated dose toxicity: dermal

Administrative data

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

Recovery period of the satellite groups was 14 days instead of 28 days. Some parameters of clinical biochemistry are either not shown or missing (ornithine decarboxylase and gamma glutamyl transpeptidase). Results of the measured body weights and urinalysis were not shown. Results of organ weights were not shown in tabular form and not all weighed organs were specified. A histopathological examination of the aorta is missing. Brain was histopathologically examined, but the sections of brain were not specified. Lesions observed in organs were not specified. Percentage of body surface area that was covered was not given. Acclimation period and housing conditions (individually or group housing) were not specified.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

modifications for dosage, biochemical parameters, and histologic evaluation. No measurement of ornithine decarboxylase and gamma glutamyl transpeptidase. 14-day recovery period in satellite groups instead of 28 days.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Zinc oxide

EC Number:

215-222-5

EC Name:

Zinc oxide

Cas Number:

1314-13-2

Molecular formula:

ZnO

IUPAC Name:

oxozinc

Test material form:

solid: crystalline

Details on test material:

not specified

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot number of test material: Sumitomo Osaka Cement Co Ltd, Osaka, Japan; 141319

INFORMATION ON NANOMATERIALS
The test material was characterised by the authors.
- Particle size & distribution: 20 nm; average size: of 29±3 nm (ZnO(SM20(-)), analysed by SEM); sperical in shape
- zeta potential of ZnO(SM20(-)): -44.4±1.0 mV; negative surface charge

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: obtained from an inhouse animal facility
- Age at study initiation: 6 weeks old
- Weight at study initiation: 150-210 g
- Housing: animals were kept in stainless steel cages
- Diet (ad libitum): rodent food; supplier: Cargill Agri Purina Inc., Kyunggido, Republic of Korea
- Water (ad libitum): reverse osmosis water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.8 ± 1
- Humidity (%): 50.8 ± 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Type of coverage:

semiocclusive

Vehicle:

other:

Remarks:

HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)-citrate buffer

Details on exposure:

TEST SITE
- Area of exposure: about 4 cm × 5 cm area on the back; approx. 10% of the total body surface area
- % coverage: not specified
- Type of wrap if used: non-irritant tape, ie, Tegaderm™ (3M) and Coban™ (3M).
- Time intervals for clipplings: once a week

REMOVAL OF TEST SUBSTANCE
- Washing: with sterile water
- Time after start of exposure: 6 hours

TEST MATERIAL
- For solids, paste formed: no
- The sterile gauze was soaked in the ZnO(SM20(-)) solution and fixed with non-irritant tape.

VEHICLE
- Justification for use and choice of vehicle (if other than water): citrate was used as coating reagent to modify the surface charge of ZnO(SM20(-)) for (-) charge.

USE OF RESTRAINERS FOR PREVENTING INGESTION: no

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 males / 10 females for main study; 5 males / 5 females for recovery study

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: a maximum dose of 1,000 mg/kg was used in this study, with 500 mg/kg as an intermediate dose and 250 mg/kg as a low dose, because significant toxicity was observed at doses of 2,000 mg/kg in an unpublished study of 14-day repeated-dose toxicity in rats.
- Fasting period before blood sampling for clinical biochemistry: 18 hours
- Post-exposure recovery period in satellite groups: 14 days

Positive control:

not specified

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily during the 90 days of treatment, and during the 14-day recovery period.

DETAILED CLINICAL OBSERVATIONS: No data

DERMAL IRRITATION: Yes
- Time schedule for examinations: daily during the 90 days of treatment, and during the 14-day recovery period.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before treatment and during the last week of the experiment.
- Dose groups that were examined: high dose group
- Parameters examined: fundus oculi

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on study termination, before necropsy.
- Anaesthetic used for blood collection: Yes, with isoflurane.
- Animals fasted: Yes, fasted for 18 hours.
- How many animals: all animals of the main study and recovery study.
- Parameters examined: white blood cell count, differential counts (neutrophils, lymphocytes, monocytes, eosinophils, basophils), red blood cell count, haemoglobin, haematocrit, mean cell volume, mean corpuscular haemoglobin, mean cell haemoglobin, mean cell haemoglobin concentration, and platelet count; analyses of reticulocyte ratio (all animals) by staining and microscopy techniques; prothrombin time and activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on study termination, before necropsy.
- Animals fasted: Yes, fastet for 18 hours.
- How many animals: all animals of the main study and recovery study.
- Parameters examined: total protein, albumin, albumin/globulin ratio, total bilirubin, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, creatinine, blood urea nitrogen, total cholesterol, triglycerides, blood glucose, calcium, inorganic phosphorus, creatine kinase, sodium, potassium, and chlorine.

URINALYSIS: Yes
- Time schedule for collection of urine: during the last week of the study.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Dose groups that were examined: high dose group
- Parameters examined: specific gravity, pH, leukocyte count, nitrite, protein, glucose, ketones, urobilinogen, bilirubin, and blood in urine. Urinary sediments were analyzed from a fresh 3-hour collection of urine, and the amount of urine was measured from the 24-hour collected urine from rats of the control and high dose group.

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
After exsanguination, the external surface, all orifices, the cranial cavity, and the thoracic and abdominal cavities and their contents were visually observed for any signs of gross abnormality.

ORGAN WEIGHT:
Absolute and relative organ weights (organ weight/body weight) were measured for each rat. For paired organs, the sum of those organs was used.

HISTOPATHOLOGY: Yes
After collection, the organs were preserved in 10% phosphate-buffered formalin solution, with the testes preserved in Davidson’s solution in preparation for histopathologic examination. Organs collected included the skin, lymph nodes, salivary glands (submandibular), bone, bone marrow (sternum, femur), thymus, trachea, bronchus, lungs, heart, thyroid gland, parathyroid glands, tongue, esophagus, stomach, small intestine, large intestine, liver, spleen, pancreas, kidneys, adrenal glands, bladder, seminal vesicles, prostate gland, testes, epididymis, ovaries, uterus, vagina, brain, pituitary gland, spinal cord, eyes, sciatic nerve, and skeletal muscle.

TISSUE DISTRIBUTION of ZnO in the PLASMA, ORGANS and FECES:
- Time schedule: blood samples and feces were collected before necropsy and the organs after necropsy.
- Parameters investigated: analyse of ZnO concentrations in blood samples, feces and the following organs: brain, liver, kidneys, testes (or ovaries), spleen, lung, stomach, small intestine, large intestine, and skin; by using ICP-AES.

Statistics:

The data obtained for body weight, food and water consumption, hematology and biochemistry, and organ weight were evaluated by one-way analysis of variance after Levene’s test to examine the homogeneity of their variance. If the data were determined to be homogeneous, the data were analyzed using Scheffe’s multiple comparison test, and if not homogeneous, by Dunnett’s T3 multiple comparison test. The data were analyzed using Statistical Package for the Social Sciences software version 12.0 (SPSS Inc, Chicago, IL, USA), and differences were considered to be statistically significant at P-values less than 0.05.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Dermal irritation:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

not specified

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

CLINICAL SIGNS:
- no dose-dependent influence in clinical signs was observed.
- Differences in clinical signs of test item-treated animals and vehicle control animals were recorded in the main study:
Formation of skin crusts observed at first in the 1000 mg/kg group 2 days after initiation of ZnO(SM20(-)), and all groups showed crust formation in a dose-dependent manner. Skin crusts also appeared in the vehicle control group, but resolved after 7 days. Skin crusts from male rats in the 1000 mg/kg group were biopsied, and found to contain areas of hyperkeratosis and papillomatosis when compared with the normal skin of controls. Scar tissue developed at the application site in one female rat from the 1000 mg/kg group.

MORTALITY:
- no death occured.

BODY WEIGHT AND WEIGHT CHANGES:
- no difference in body weight was observed between the experimental and control groups.

FOOD CONSUMPTION:
- no dose-dependent influence in food consumption was observed. The observed changes were considered temporary, given that body weight did not change significantly:
main study: food intake was significantly decreased in male rats from the 250 and 500 mg/kg groups in comparison with that in the control group at week 2. A decrease was also observed in female rats from the 250 mg/kg group at week 1 in comparison with the control group, and a increase was seen in female rats from the 1000 mg/kg group at week 2 in comparison with the control group.
recovery study: food intake was decreased in male rats form the 1000 mg/kg group after week 2 of recovery.

WATER CONSUMPTION:
- no dose-dependent influence in food consumption was observed. The observed changes were considered temporary, given that body weight did not change significantly:
main study: male rats from the 1000 mg/kg group consumed less water than the control group at week 11.
recovery study: water consumption increased significantly in female rats from the 1000 mg/kg group at weeks 1 and 2.

OPHTHALMOLOGICAL FINDINGS:
- no significant changes were observed on ophthalmologic examination.

HAEMATOLOGICAL FINDINGS:
- no dose-dependent influence in haematology was observed and the results were not consistent between male and female rats.
- Statistically significant differences in haematological parameters of test item-treated animals with the control animals were recorded (not dose-dependent findings):
main study: decreased number of lymphocytes was documented in male rats from the 1000 mg/kg group in comparison with the control group (P<0.05). Increased mean corpuscular haemoglobin was observed in female rats in comparison with the vehicle control group (P<0.05).

CLINICAL BIOCHEMISTRY FINDINGS:
- no dose-dependent influence in clinical biochemistry was observed and the results were not consistent between male and female rats.
- Statistically significant differences in biochemical parameters of test item-treated animals with the control animals were recorded (not dose-dependent findings):
recovery study: aspartate aminotransferase and creatine kinase levels were significantly decreased in male rats (P<0.01) from the 1000 mg/kg group in comparison with the control group. The albumin/globulin ratio in female rats was significantly increased in comparison with the vehicle control group (P<0.05).

URINALYSIS FINDINGS:
- no significant changes were observed in urinylsis.

ORGAN WEIGHT FINDINGS INCLUDING ORGAN / BODY WEIGHT RATIOS:
- no dose-dependent influence in organ weights was observed and the changes in organ weights occured sporadically.
- Statistically significant differences in organ weights of test item-treated animals compared to the control animals were recorded (not dose-dependent findings):
main study: significantly decreased (P<0.05) weight of the epididymis was observed in male rats from the 500 mg/kg group.
Recovery study: weight of the thymus decreased significantly in male rats from the 1000 mg/kg group in comparison with the control group (P<0.05).
- There was no significant difference in organ weight for female rats in comparison with the control group.

GROSS PATHOLOGICAL FINDINGS:
- no dose-dependent influence in gross pathological findings was observed and the abnormalities were also observed in the control group.
- Abnormalities in the internal necropsy findings of test item-treated animals and control animals were recorded (not dose-dependent findings) in the main study:
- 250 mg/kg: a reddish stomach nodule was found in two female rats.
- 500 mg/kg: atrophy of the right seminal vesicle was observed in one male rat. A reddish nodule in the stomach, a reddish color change in the caudal lobe of the liver, a light brown color change in the right kidney, and fragility of the right adrenal gland were observed in female rats.
- 1000 mg/kg: one red nodule and a yellow mass in the stomach, a light brown color change of the right kidney, and a reddish color change in the accessory lobe of the liver were documented in female rats.
- Vehicle control group: right prostate gland hyperplasia and a yellowish mass in the head of the right epididymis was observed in one male rat each.
- Control group: red nodule in the stomach (two cases) and a light brown change in the right kidney color (one case) occurred in female rats.
- No abnormalities in the internal gross pathology were observed in the other animals of the test item-treated and control groups.
- No abnormalities were observed in the external gross pathology of all animals in each group.

HISTOPATHOLOGICAL FINDINGS:
- no test item-related influence observed in histopathological findings. Also, lesions in other organs were considered as nonspecific.
- Changes in histopathology of test item-treated animals and control animals were recorded (not test item-related findings) in the main study:
- 1000 mg/kg and control: areas of necrosis with regenerative hyperplasia and pigmentation were found in the livers of female rats.

TISSUE DISTRIBUTION of ZnO nanoparticles:
- no differences in ZnO NP concentration were seen between females and males, with higher concentrations found in the liver, large intestine, small intestine, and feces upon increasing doses when compared with the negative and vehicle control groups. However, ZnO NP concentrations remained the same in the brain, testes, ovaries, spleen, stomach, plasma, kidneys, and lung as in the negative and vehicle control groups.
- The test material seemed to remain on the skin after washing was done with distilled water prior to necropsy.

Effect levels

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In this dermal RDT study, ZnO nanoparticles with a negative surface charges at doses of 250, 500 mg/kg, and 1000 mg/kg were repeatedly administered by dermal administration for 90 days in SD rats. The toxicity of these NPs in target organs was evaluated, but there was no study-related internal organ toxicity. No effects were observed in mortality, ophthalmology and urinalysis, and no test item-related effects were observed in body weights, food and water consumption, haematology, clinical biochemistry, gross pathology and histopathology. A temporary, dose-dependent inflammation of the skin was observed at the application site. Therefore, no adverse effects were observed with ZnO nanoparticles (20 nm, negatively charged) up to 1000 mg/kg body weight in both sexes of rats and this dose level represents the NOAEL.

The results of this dermal 90-day RDT study in SD rats can generally be regarded as reliable with restrictions, because the study was conducted based on the OECD guideline 411 and according to GLP. The methods and results are described appropriately, and the conclusions are plausible.
However, the recovery period of the satellite groups was 14 days instead of 28 days. Furthermore, not all data (e.g. urinalysis) are shown in the publication. Some parameters of clinical biochemistry are either not shown (total bilirubin, albumin, glucose, total cholesterol, triglycerides, sodium, calcium, potassium, chlorine, inorganic phosphorus, alkaline phosphatase) or missing (ornithine decarboxylase and gamma glutamyl transpeptidase).
Additionally, the results of the measured body weights and urinalysis were not shown in the publication. The organ weights of the animals were measured and the relative organ weights were calculated, but they were not shown in tabular form in the results. In the results, the authors said that the organ weights of the thymus and epididymis were decreased in the males of the high-dose group, but it was not specified whether these were absolute or relative organ weights. In addition, it is unclear which other organs were weighed.
In addition, a histopathological examination of the aorta is missing. The brain was histopathologically examined, but it was not specified which sections of brain were examined. Therefore, it is unclear whether the sections of medulla/pons, cerebellar cortex and cerebral cortex were included. Furthermore, the authors said that in addition to the necrosis areas with regenerative hyperplasia and pigmentation in the livers of female rats in the control group and in the high-dose group, lesions were also found in other organs. They considered these lesions as unspecific, but in which organs the lesions were found was not specified.
Furthermore, the percentage of body surface area that was covered was not given. The acclimation period and the housing conditions were not specified, so it is unclear whether the animals were kept individually or in groups.
Therefore, the study is judged as reliable with restrictions because it represents a guideline study without detailed documentation.