Zinc oxide

Acute Toxicity: other routes

Administrative data

Endpoint:

acute toxicity: other routes

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD guideline 407: Repeated dose 28-day oral toxicity study in rodents

Deviations:

yes

Remarks:

, single i. v. application, 4 weeks observation period

Principles of method if other than guideline:

The aim of this study was to investigate the comparative toxicity of Zinc oxide as coated or uncoated nanoparticles, as a pigment form or as Zinc sulfate. The intravenous application route represents a worst-case scenario and was selected to guarantee systemic bioavailability of 100%. The complete study consisted of a 28-day toxicity study part in male and female rats (subset 0) as well as an organ distribution part in male rats after 1 and 29 days after dosing (subsets 1 and 2). The objective to examine organ distribution histopathologically was omitted as no adverse or persistent effect was observed after the observation period of up to 4 weeks. Zinc oxide (nanoscale, coated), Zinc oxide (nanoscale, uncoated), Zinc oxide (pigment) and
Zinc sulfate were tested in parallel at equimolar dosages.

GLP compliance:

yes (incl. QA statement)

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: nano- and micromaterial

Details on test material:

1. Zinc oxide (nanoscale, coated):
- Name of test substance: Z-Cote HP1
- Synonym: Zinc oxide (nanoscale, coated with triethoxyoctylsilane)
- CAS No: 1314-13-2
- Batch identification: CNGB1602
- Purity: Zinc oxide >97.1%, triethoxyoctylsilane 3.4%
- Homogeneity: given
- Stability: stable until 15 Feb 2010
- Physical state/color: solid/white
- Particle structure and size: Z-COTE HP 1 analysis of Jun 2007: Median, quantity (d 0.5) = 68 nm, Median, Volume (d 0.5) = 140 nm
- Storage conditions: room temperature

2. Zinc oxide (nanoscale, uncoated):
- Name of test substance: Z-Cote
- Synonym: Zinc oxide (nanoscale, uncoated)
- CAS No: 1314-13-2
- Batch identification: EHFA0907
- Purity: no details
- Homogeneity: given
- Stability: Stable until 01 Jan 2010
- Physical state/color: solid/white
- Particle structure and size: Z-COTE analysis of Jan 2009: Median, quantity (d 0.5) = 67 nm; Median, Volume (d 0.5) = 131 nm
- Storage conditions: room temperature

3. Zinc oxide (pigment):
The test substance was used with the given specifications of the producer. No further analyses were conducted.
- Name of test substance Zinc oxide, powder
- Synonym: Zinc oxide (pigment)
- CAS No: 1314-13-2
- Batch identification: S35583-206
- Purity: >99%
- Homogeneity: given
- Stability: stable until 30 Sep 2009
- Physical state/color: solid/white
- Particle structure and size: Information provided by the sponsor: <1μm
- Storage conditions: room temperature

4. Zinc sulfate:
The test substance was used with the given specifications of the producer. No further analyses were conducted.
- Name of test substance: Zinc sulfate × 7 H2O 99%
- Synonym: Zinc sulfate (ZnSO4); Zinc sulfate heptahydrate
- CAS No: 7446-20-0
- Batch identification: 02616TC-126
- Purity: 100%
- Homogeneity: given
- Stability: stable until 01 Sep 2009
- Physical state/color: solid/white
- Storage conditions: room temperature

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld/Germany
- Age at start of the study: 6 weeks
- Weight at study initiation: males' group means: 162.4 - 179.6 g; females' group means: 137.4 - 143.4 g
- Fasting period before study: 16 - 20 h
- Housing: individually, dust free bedding supplied by Type Lignocel PS 14 fibres
- Individual metabolism cages: yes
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 10, fully airconsitioned
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Administration / exposure

Route of administration:

intravenous

Vehicle:

other: 0.9% saline (zinc sulfate), calf serum (nanoscaled coated and uncoated, ZnO pigment)

Details on exposure:

I.V. APPLICATION:
- Reason for the selection of the route of administration: The route of administration was chosen to obtain information on the toxicity and tissue distribution after a worst case exposure scenario with bioavailability of 100%.

PREPARATION OF DOSING SOLUTIONS:
- Rate of preparation: the test substance preparations was prepared on the day of the administration (nanoscaled coated, microscaled) or on the day before (nanoscaled coated)
- Preparation: All preparations were kept homogenous during application by a magnetic stirrer. NANOSCALED COATED: The test substance was weighed and filled up with calf serum to the desired volume. This preparation was homogenized by an ultrasonic stirrer and put in the ultra sonic bath for 5 minutes. Then the preparation was kept on a magnetic stirrer for about 20 h and thereafter homogenized once again in the ultra sonic bath. NANOSCALED UNCOATED and ZnO PIGMENT: The test substance was weighed up and filled up with calf serum to the desired volume. This preparation was homogenized 5 minutes in an ultra sonic bath. Zn SULFATE: The test substance was weighed and filled up with 0.9% saline to the desired volume and diluted.

HOMOGENEITY AND STABILITY OF TEST MATERIAL: All preparations were kept homogenous during application by a magnetic stirrer.

Doses:

subset 0: 1 and 5 mg/kg bw nanoscaled coated and uncoated, 5 mg/kg bw ZnO pigment, 4 x 4.4 mg/kg bw Zinc sulfate
subset 1: 1 and 5 mg/kg bw nanoscaled coated and uncoated, 5 mg/kg bw ZnO pigment, 17.6 mg/kg bw Zinc sulfate
subset 2: 1 and 5 mg/kg bw nanoscaled coated and uncoated, 5 mg/kg bw ZnO pigment, 4 x 4.4 mg/kg bw Zinc sulfate

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 28 days
- Frequency of observations and weighing: daily (clinical observation), weekly weighing
- Necropsy of survivors performed: yes
- Other examinations performed: food and water consumption

URINALYSIS: pH, protein, glucose, ketones, urobilinogen, blood, specific gravity, sediment, color, turbidity, volume

CARDIAC BIOMARKERS:
Wang et al (2007) and Liu et al (2009) described the increase of serum lactate dehydrogenase (LDH), α-hydroxybutyrate dehydrogenase (α-HBDH) and creatine kinase (CK) levels as biomarkers of myocardiotoxicity after oral and intraperitoneal administration of titanium dioxide nanoparticles in mice. The mentioned parameters are regarded as being not specific for cardiotoxicity in humans as well as animals (Cunningham et al, 2002;
O’Brien, 2008). In order to address the issue of potential cardiotoxicity with an up-to-date clinical pathology parameter cardiac troponin I measured was measured in this study (Wallace et al, 2004).

ACUTE PHASE PROTEINS:
In rats, α2-macroglobulin and haptoglobin are regarded as major acute phase proteins (Eckersall, 2009). Haptoglobin was measured in serum samples with a photometric assay, α2-macroglobulin with an ELISA system.

HEMATOLOGY: leucocyte count, erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume/hemoglobin/hemoglobin concentration, platelet count, differential blood count, reticulocytes, prothrombin time

CLINICAL CHEMISTRY:
Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, γ-Glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium

DETAILED CLINICAL OBSERVATION were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high). The following parameters were examined:

1. abnormal behavior during “handling”
2. fur
3. skin
4. posture
5. salivation
6. respiration
7. activity/arousal level
8. tremors
9. convulsions
10. abnormal movements
11. impairment of gait
12. lacrimation
13. palpebral closure
14. exophthalmus
15. feces (appearance/consistency)
16. urine
17. pupil size

PATHOLOGY:
organ weights: Anesthetized animals, kidney, liver, adrenal gland, testes, Epididymides, ovary, uterus, spleen, brain, heart, thymus, thyroid glands

HISTOLOGY:
gross lesions, brain, lung, liver, spleen, kidney

Statistics:

body weight, body weight change, food consumption:
A comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means. A comparison of the dose group with the control group was performed using the Welch t-test (two-sided) for the hypothesis of equal means.

Results and discussion

Effect levels

Sex:

male/female

Dose descriptor:

other: differential blood cell count and clinical chemistry parameters (indicative for impairment of liver/kidney function)

Remarks on result:

other: comparative toxicity study of Zinc oxide as coated or uncoated nanoparticles, as a pigment form or as Zinc sulfate showed no effect at the end of the observation period of 4 weeks, when injected intravenously at dose levels of 1 or 5 mg/kg bw

Mortality:

not observed

Clinical signs:

No test substance-related findings were observed.

Body weight:

no effects

Other findings:

Subset 0: No test substance-related findings were observed.

Subset 1:
The absolute monocyte counts were increased in the groups treated with 5 mg/kg nanoscaled coated ZnO and 1 mg/kg nanoscaled uncoated ZnO. The absolute and relative large unstained cell (LUC) counts were significantly increased in the group treated with 5 mg/kg nanoscaled coated ZnO and the relative LUC counts were increased in the ZnO pigment treated group.
In rats, which got the test items at study day 28 (test groups 10-16), significant increases were measured with regard to urea, creatinine, calcium and magnesium levels as well as higher alkaline phosphatase (ALP) activity in test group 16 (zinc sulfate). In test group 15 (zinc oxide [pigment]) the AST and ALP activities were significantly increased. In test group 12 (zinc oxide [nanoscale, coated]; 5 mg/kg bw) the AST and ALP activities as well as the bilirubin and inorganic phosphate concentrations were statistically significantly higher compared to controls.
The haptoglobin values were statistically significantly increased in rats of test groups 11 and 12 (zinc oxide [nanoscale, coated]; 1 and 5 mg/kg bw) and of test group 15 (zinc oxide [pigment]).

Subset 2: No test substance-related findings were observed.

Any other information on results incl. tables

Subset 0: No test substance-related findings were observed.

Subset 1: The absolute monocyte counts were increased in the groups treated with 5 mg/kg nanoscaled coated ZnO and 1 mg/kg nanoscaled uncoated ZnO. The absolute and relative large unstained cell (LUC) counts were significantly increased in the group treated with 5 mg/kg nanoscaled coated ZnO and the relative LUC counts were increased in the ZnO pigment treated group. In rats, which got the test items at study day 28 (test groups 10-16), significant increases were measured with regard to urea, creatinine, calcium and magnesium levels as well as higher alkaline phosphatase (ALP) activity in test group 16 (zinc sulfate). In test group 15 (zinc oxide [pigment]) the AST and ALP activities were significantly increased. In test group 12 (zinc oxide [nanoscale, coated]; 5 mg/kg bw) the AST and ALP activities as well as the bilirubin and inorganic phosphate concentrations were statistically significantly higher compared to controls. The haptoglobin values were statistically significantly increased in rats of test groups 11 and 12 (zinc oxide [nanoscale, coated]; 1 and 5 mg/kg bw) and of test group 15 (zinc oxide [pigment]).

Subset 2: No test substance-related findings were observed.

Applicant's summary and conclusion

Executive summary:

The comparative toxicity study of Zinc oxide as coated or uncoated nanoparticles, as a pigment form or as Zinc sulfate showed no effect at the end of the observation period of 4 weeks, when injected intravenously at dose levels of 1 or 5 mg/kg bw. Therefore, no further examinations in the perfusion-fixed animals of subset 2 were performed. Very mild deviations in the differential blood cell count (monocyte, large unstained cells) and few clinical chemistry parameters, indicative for a minimal impairment of liver function (Zinc oxide preparations) or of kidney function in addition (Zinc sulfate) were observed 1 day after intravenous injection of 5 mg/kg bw pigmented or coated nanoscale Zinc oxide or an equimolar Zinc sulfate dose. At 1 mg/kg bw no biological relevant effect was observed. Among the sensitive markers of acute phase reactions haptaglobulin was increased, while α2-macroglobulin was not affected. Finally, the comparative screening showed only minor and in any case transient effects without toxicological relevance. There was no biologically relevant difference in the injected form of Zinc oxide, especially no enhancement of the observed effects due to the injection of nanoscale material. The equimolar injection of the Zinc ion as Zinc sulfate led qualitatively and quantitatively to a slight enhancement of the observed variation in homoeostasis. In any case, 4 weeks after treatment there was virtually no difference in comparison to the respective control group, irrespectively of the injected dose level, the usage of Zinc oxide as coated or uncoated nanoparticles, as pigment or as Zinc sulfate.