Zinc oxide

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not specified

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Results are not well documented because general endpoints for evaluation of toxicity were included in the study design but not reported in detail in the publication. Test substance is poorly described. It was purchased in the form of a water dispersion, but the purity of the solution used and the ZnO concentration in the dispersion were not specified. Stability, homogeneity and the concentrations of the test substance in the formulations which the animals received were not analysed. Results of clinical observations were not given. Detailed clinical and functional observations or gross pathological examinations were not performed. No analysis of urea in blood. Red blood cells, total and differential white blood cell levels were not shown in results. Results of urinalysis was not shown. Organ weight of the uterus and the epididymides was missing. No histopathological examination of the aorta and on a section of bone marrow was performed. It is unclear whether the Preyer`s patches were included in the histopathological examination of the small and large intestines. It is unclear whether the cervical or lumbar areas of the spinal cord were included at the histopathological examination. The initial body weight of the test animals was missing.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

No data of clinical sings. Detailed clinical & functional observations & gross pathology were not performed. No histopathological examination of aorta & bone marrow. No initial body weight of animals were given. No analysis of urea in blood.

GLP compliance:

yes

Remarks:

good laboratory practices (GLP) for toxicity test guidance on drugs (Notification No. 2005-60, Oct. 21, 2005) issued by the Korea Food and Drug Administration.

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:

other:

Remarks:

ZnO nanoparticles in form of a water dispersion

Details on test material:

Source: Nanostructured & Amorphous Materials, Inc. (TX, USA)
primary size: 40 nm
surface area (BET): 60 ± 10 m²/g

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Nanostructured & Amorphous Materials, Inc. (TX, USA)

INFORMATION ON NANOMATERIALS
- Chemical Composition: ZnO
- Hydrodynamic size: 201.75 ± 17.15 nm (in distilled water)
- Zeta potential: ZnO nanoparticle: negatively charged at neutral and basic pH; positively charged at pH < 4

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: OrientBio Ltd, Seongnam, Korea
- Age at study initiation: 8 weeks old
- Housing: animals were separated by sex and kept in polycarbonate cages
- Diet (ad libitum): gamma-irradiated rodent diet; source: LabDiet 5002; PMI Nutrition, Richmond, USA
- Water (ad libitum): autoclaved water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 – 24
- Humidity (%): 45 - 60
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

not specified

Vehicle:

water

Remarks:

distilled

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- Administration volume: 10 mL/kg bw/day

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

67.1 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

134.2 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

268.4 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

536.8 mg/kg bw/day (nominal)

No. of animals per sex per dose:

11 males / 11 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of a 14-day repeated toxicity study.

Positive control:

not specified

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule (clinical signs): daily
- Time schedule (mortality): daily

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during the study period

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 during the study period

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during the last week of treatment
- Dose groups that were examined: treatment groups
- Parameters examined: external eye, ocular fundus, conjunctiva, sclera, cornea, lens and iris by an ophthalmoscope.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day 91 (males) and day 92 (females)
- Anaesthetic used for blood collection: Yes, anesthetized with ether.
- Animals fasted: not specified
- How many animals: all animals
- Parameters examined: red blood cells (RBC), haemoglobin concentration, haematocrit, (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelets, white blood cells (WBC) and differential WBC.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day 91 (males) and day 92 (females).
- Animals fasted: not specified
- How many animals: all animals
- Parameters examined: blood urea nitrogen (BUN), total cholesterol, total protein, albumin (TB), albumin/globulin ratio (A/G ratio), total bilirubin, alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyltransferase (GGT), creatinine (CRE), triglycerides (TG), glucose (GLU), sodium (Na), potassium (K), chloride (Cl), calcium (Ca) and phosphorus (P).

URINALYSIS: Yes
- Time schedule for collection of urine: during the last week of treatment
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: not specified
- Parameters examined: pH, specific gravity, number of leukocytes, nitrite levels, protein levels, ketone bodies, urobilinogen concentration, bilirubin concentration, glucose concentration and occult blood content.

NEUROBEHAVIOURAL EXAMINATION: Not specified
IMMUNOLOGY: Not specified

Sacrifice and pathology:

GROSS PATHOLOGY: Not specified

ORGAN WEIGHT:
At necropsy, the heart, liver, lung, spleen, thymus, kidney, adrenal gland, testes, ovary, brain and pituitary gland of each animal was removed and weighed.

HISTOPATHOLOGY: Yes
The nasal cavity, eyes with Harderian glands, spinal cord (thoracic portion together with corresponding vertebral bones), salivary glands, stomach, small intestine (duodenum, jejunum and ileum), large intestine (cecum, colon and rectum), pancreas, urinary bladder, skin with mammary glands, mesenteric lymph nodes, trachea, oesophagus, thyroid glands, tongue, thigh muscle, sciatic nerve, epididymides, seminal vesicles, prostate (ventral and dorsolateral lobes), uterus, ovaries and vagina were removed. Tissues were fixed in 10% neutral buffered formalin, except for the testes and epididymides, which were fixed in Bouin’s solution and the eyes with Harderian glands, which were fixed in Davidson’s solution. All tissues were routinely processed for paraffin embedding, sectioning, and hematoxylin and eosin staining. All tissues from the control and the treatment groups underwent histological assessment.

Statistics:

For the sub-chronic toxicity study and in vitro tests, except for the urinalysis and histopathological findings, data were analysed using one-way ANOVA (TDMS ver.4.0; KFDA, Osong, Korea). When statistically significant differences were indicated (P<0.05), Dunnett’s t-test was employed to compare control and treatment groups. In the case of histopathological changes, incidences were compared using the Poly-3 test.

Results and discussion

Results of examinations

Clinical signs:

not specified

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 536.8 mg/kg bw/day: significant (P<0.05) dose-related decreases in male rats

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:

effects observed, treatment-related

Description (incidence and severity):

- 536,8 mg/kg bw/day: male rats showed significant decreases (p<0.01) in the levels of Hb, HCT and MCH, whereas the number of platelets (PLT) was increased (p<0.01) compared to the control group. Significant decrease (p<0.01) in the levels of MCV and MCH in female rats compared to the control group.

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:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- 536,8 mg/kg bw/day: 6/11 (55%) males and 7/11 (66%) females showed mild to moderate pancreatitis with focal lymphocyte infiltration and mild acinar apoptosis.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Details on results:

MORTALITY:
- no death occured.

BODY WEIGHT WEIGHT CHANGES:
- no significant dose-related changes in the body weight of female rats

FOOD CONSUMPTION:
- There were no significant differences in food consumption between the treatment groups and the control group were observed.

WATER CONSUMPTION:
- no significant differences in water intake between the treatment groups and the control group were observed.

OPHTHALMOLOGICAL FINDINGS:
- no significant differences between the treatment and control groups were observed.

HAEMATOLOGICAL FINDINGS:
- there were no significant differences between the treatment and control groups in the coagulation assay.
- 536,8 mg/kg bw/day: significant increase in the number of platelets in female rats compared to the control group (p<0.01).
- 134.2 and 536.8 mg/kg bw/day: a significant decrease were observed in males in the 134.2 mg/kg bw/day group (p<0.05) and 536.8 mg/kg bw/day group (p<0.01) compared to the control group.

CLINICAL BIOCHEMISTRY FINDINGS:
- 536,8 mg/kg bw/day: levels of ALP and phosphorus in males were significantly higher (p<0.01) than the levels in vehicle controls. Significantly lower levels of albumin in females compared to the control females were observed.
- 67.1, 134.2, 268.4 and 536.8 mg/kg bw/day: significant increase in sodium levels in females in all dose groups compared to the controls, while in the males it was only observed in the 268.4 and 536.8 mg/kg bw/day group.
- 134.2, 268.4 and 536.8 mg/kg bw/day: the chloride levels were significantly increased in females in the three dose groups, and in the males of the highest dose group. Significantly lower levels of total protein in both sexes in the 536.8 mg/kg bw/day group compared to the vehicle control and it was significantly decreased in amles in the 268.4 mg/kg bw/day group and in females in the 134.2 mg/kg bw/day (p<0.05).
- 67.1 mg/kg bw/day: triglyceride levels were significantly incereased in females compared to the vehicle control (p<0.05).

URINALYSIS:
- no significant differences between the treatment and control groups were observed.

ORGAN WEIGHT FINDINGS INCLUDING ORGAN / BODY WEIGHT RATIOS:
- no significant organ-weight changes were observed in either sex treated with ZnO NPs after 90 days (data not shown).

HISTOPATHOLOGICAL FINDINGS:
- other treatment groups (67.1 - 268.4 mg/kg bw/day group) had no significant pathological changes compared with the vehicle control group. There were no significant pathological changes in other organs (data not shown).

BIOPERSISTENCE OF ZnO NANOPARTICLES:
- the white, milky-colored ZnO NP solution cleared within a few minutes when added to acidic artificial gastric fluid (AGF, pH 1.7). After 24 h, around 98% of the ZnO NP mass had dissolved in the AGF, whereas ZnO NPs in DW showed minimal dissolution. After a 24-h incubation of ZnO NPs in AGF, increasing the pH of the solution to 7.4 did not result in reaggregation. Likewise, ZnO NPs in DW or RPMI-1640 culture medium supplemented with 10% foetal bovine serum (FBS) maintained their morphology and size. However, ZnO NPs in AGF dissolved fully; only the grid could be observed.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

268.4 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

2

Dose descriptor:

LOAEL

Effect level:

536.8 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In this 90-day RDT study, ZnO nanoparticles at doses of 67.1, 134.2, 268.4 or 536.8 mg/kg bw/d were repeatedly administered by gavage in SD rats for 90 days. The mean body weight gain in males given 536.8 mg/kg ZnO nanoparticles was significantly lower than that of control male rats, whereas no significant differences were observed between the other treatment groups and the controls. Male and female rats dosed at 536.8 mg/kg ZnO nanoparticles had significant changes in anaemia-related haematologic parameters. Mild to moderate pancreatitis also developed in both sexes dosed at 536.8 mg/kg, whereas no histological changes were observed in the other treatment groups.
According to the authors, based on the findings of this 13-week RDT study using ZnO nanoparticles, the NOAEL in SD rats was 268.4 mg/kg with the highest dose level of 536.8 mg/kg bw/d representing the LOAEL.

The results of this oral 90-day RDT study in SD rats support the evidence that ZnO NPs up to the dose of 268.4 mg/kg bw/d did not cause general toxicity in SD rats under the conditions of this study. The identified target organs of toxicity are comparable to those observed in the studies conducted by Park et al 2014a and 2014b as well as Kim et al. 2014.

However, although the study was conducted to a relevant OECD guideline and the methods used meet generally accepted scientific principles for assessment, the results are not well documented because general endpoints for evaluation of toxicity were included in the study design but not reported in detail in the publication. Since the test substance is poorly described. It was purchased in the form of a water dispersion, but the purity of the solution used and the ZnO concentration in the dispersion were not specified. The stability and dissolution of the test substance were analysed only in artificial gastric fluid. However, the stability, homogeneity and the concentrations of the test substance in the formulations which the animals received were not analyzed.
Clinical observations have been made during the study, but no information is provided in the results. Furthermore, detailed clinical observations and functional observations were not performed. In addition, there is no information that a gross pathological examination was carried out. No analysis of urea in blood was performed. During the haematological examination, the content of red blood cells, total and differential white blood cells of the animals was determined, but not shown in the results.
A urinalysis was carried out, but the levels of the parameters were not shown in the results. The organ weight of the uterus and the epididymides is missing. No histopathological examination of the aorta and on a section of bone marrow was performed. It is also unclear whether the Preyer`s patches were included in the histopathological examination of the small and large intestines. The spinal cord (thoracic portion together with the corresponding vertebral bones) was investigated, but it is unclear whether the cervical or lumbar areas were also included. Furthermore, the initial body weight of the test animals is missing.
Therefore, the study is only of supportive nature and regarded as not reliable in a regulatory context.