Registration Dossier

Administrative data

Endpoint:
immunotoxicity
Remarks:
other: both in vitro and in vivo (subacute)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study is well documented and meets general and acceptable scientific principles.

Data source

Reference
Reference Type:
publication
Title:
Comparative study of respiratory tract immune toxicity induced by three sterilisation nanoparticles: Silver, zinc oxide and titanium dioxide
Author:
Liu H, Yang D, Yang H, Zhang H, Zhang W, Fang Y, Lin Z, Tian L, Lin B, Yan J and Xi Z.
Year:
2013
Bibliographic source:
Journal of Hazardous Materials, 248–249, 478– 486

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
In the in vivo study, 6 rats/group were exposed to nanoparticles by intratracheal instillation once every 2 d for 5 weeks. 24 h after the last exposure, the right lung was then lavaged with normal (physiological) saline to collect bronchoalveolar lavage fluid (BALF) in which both oxidative damage and cytokines were detected. In the in vitro study, alveolar macrophages (AMs) were exposed to the nanoparticles for 24 h and were then analyzed for morphological changes and cell viability.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: nanoform
Details on test material:
Test substance: Nano-ZnO
- Size: 19.61 ± 5.83 nm
- Density: 5.78 g/cm3
- Specific surface area: 45 m2/g
- Shape: Hexagonal
- Composition: ZnO >99.9%

Test animals

Species:
rat
Strain:
Wistar
Sex:
not specified
Details on test animals and environmental conditions:
Forty-two Wistar rats (body weight, 160–180 g) were divided randomly into: control group, and 3.5 or 17.5 mg/kg bw dosage groups. One week prior to the beginning of the experiment, the rats were housed in pairs under controlled environmental conditions (temperature 24 ± 1 ◦C, humidity 50 ± 5%, lights on 07:00–19:00 h). The treatment was performed in a Grade II animal room, and there were no other air pollutants in the environment. Rodent diet and water were provided ad libitum.

Administration / exposure

Route of administration:
other: intratracheal
Vehicle:
other: foetal bovine serum
Details on exposure:
In vivo: The rats were exposed to nanoparticles by intratracheal instillation once every 2 d for 5 weeks.
In vitro: Alveolar macrophages (AMs) underwent primary culture and were used. Specific pathogen-free adult Wistar rats (220–250 g) were provided by the Laboratory Animal Center of the Institute of Health and Environmental Medicine, Academy of Military Medical Sciences (Tianjin, China). The rats were anaesthetized with 10% chloral hydrate. They were killed by bloodletting from the abdominal aorta. The rats were sterilised for 1–2 min with 75% ethanol, and then they were moved to the clean benches. BALF was extracted using phosphate-buffered saline (PBS) as lavage fluid, and then AMs were separated. Cells were prepared. They were then cultured in RPMI 1640 medium (HyClone, Thermo Scientific, Jülich, Germany) supplemented with 10% (v/v) FBS (Gibco), penicillin (100 U/mL) and streptomycin (100 µg/mL) in a cell incubator (37⁰C, 5% CO2) and allowed to attach for 3 h. The cells were rinsed twice with PBS to remove non-adherent cells. Fresh culture medium was added to each well, and the cells were allowed to incubate for 48 h before treatment.
Duration of treatment / exposure:
In vivo: 5 weeks
In vitro: 24 h
Frequency of treatment:
In vivo: Once every 2 d
In vitro: Continous for 24 h
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 3.5 and 17.5 mg/kg bw/day (for in vivo)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 5, 10, 25, 50, and 100 µg/mL (for in vitro)
Basis:
nominal conc.
No. of animals per sex per dose:
In vivo: 6 animals/dose
In vitro: 1 × 100,000 cells/well
Control animals:
yes, concurrent vehicle
Details on study design:
In vivo:
-Detection of oxidative damage in BALF: The the levels of malondialdehyde (MDA), superoxide dismutase (SOD), reduced glutathione (GSH) and nitrogen oxide (NO) in BALF were detected to determine the oxidative damage to the lung tissue of rats by nanoparticles. The rats were killed by bloodletting from the abdominal aorta under anaesthesia with ether 24 h after the last exposure to nanoparticles. The right lung was then lavaged with normal (physiological) saline at 37⁰C to collect BALF. BALF was centrifuged at 2500 rpm for 15 min, and the supernatants were collected. The levels of GSH, SOD, MDA and NO were measured using reagent kits purchased from Jiancheng Bioengineering (Nanjing, China).
-Detection of cytokines in BALF: The levels of interleukin (IL)-1, IL-6, tumour necrosis factor-alpha (TNF-) and macrophage inflammatory protein (MIP-2) in BALF were detected using the double-antibody sandwich ELISA method to determine the immune effects within the respiratory tract of rats after exposure to nanoparticles. The levels of these cytokines were measured using reagent kits purchased from R&D Systems (Minneapolis, MN, USA). Optical density was measured using an Automatic Multifunction Microplate Reader at 450 nm.

In vitro:
-Morphological changes of AMs after 24 h exposure: The cells were seeded onto 96-well plates at 1.0 × 105 cells per well. They were incubated with particle suspensions (10 and 50 µg/mL) in a cell incubator for 24 h. Morphological changes in AMs were observed with an Inverted Microscope (Olympus BX51, Japan).
-Cell viability assays: Cell viability was determined using the WST-8 assay (Beyotime, China). The cells were plated onto 96-well plates at 1.0 × 105 cells per well in a 100 µL culture medium and incubated for 48 h at 37⁰C. Then, the cells were treated with 0–100 µg/mL of nanoparticle suspensions for 24 h in a cell incubator. At the end of treatment, the cell culture medium was aspirated carefully. New medium and 10 µL of WST-8 solution were added to each well, mixed thoroughly, and incubated at 37⁰C for 4 h. Subsequently, the absorbance was read on a multi-well plate Reader at 450 nm.
-LDH measurement: LDH leakage, another objective measure of cytotoxicity on the basis of membrane integrity damage, was measured using a commercial LDH kit (Jiancheng Bioengineering), according to manufacturer protocols. After incubation with nanoparticles for 24 h, the cell medium was collected and centrifuged at 3000 rpm for 10 min. Then, the supernatant was collected for measurement of LDH activity. Absorption was measured at 340 nm.
-Neutral red assay: Phagocytosis reflects the non-specific defence capability of AMs. In the present study, the effect of the phagocytosis of AMs induced by nanoparticles was determined using the neutral red assay. The cells were seeded at 4.0 × 105 cells per well in 96-well plates with 200 µL of culture medium per well. After treatment with 0–100 µg/mL of the three nanoparticles for 24 h, the cells were carefully washed twice with culture medium to wash away residual nanoparticles. Then, 200 µL of 0.1% neutral red was added to each well and incubated at 37⁰C for 3 h. The cells were washed meticulously to remove neutral red that had not been phagocytised by AMs, and freshly prepared 200 µL aliquots of cell lysate (acetic acid:ethanol was 1:1) were added to each well. Then, the cells were stored overnight, and absorption was read on a multi-well plate reader at 550 nm.

Results and discussion

Results of examinations

Details on results:
In vivo:
-Oxidative stress level in BALF: The balance of oxidants and antioxidants is very critical to healthy organisms. The levels of reduced glutathione (GSH) and superoxide dismutase (SOD) were measured to determine the antioxidative response of the respiratory tract to nanoparticles. Regarding GSH and SOD activities, all exposure groups were significantly lower than those of the control group. A dose-dependent relationship between SOD activities and the concentrations of nano-ZnO was observed. In addition, the concentrations of malondialdehyde (MDA) and nitrogen oxide (NO) were monitored to elucidate the lipid peroxidation induced by nanoparticles. The concentrations of MDA and NO in BALF in all nanoparticle groups increased significantly compared with those of the control. The elevated concentrations of MDA and NO were observed in a dose-dependent manner.
-Cytokine levels in BALF: The concentrations of TNF-α was increased significantly. MIP-2 concentrations were also increased significantly in all groups.

In vitro:
-Morphological changes in AMs after 24-h exposure: The control cells had normal morphologies with transparent cytoplasms. However, after treating with 10 or 50 µg/mL for 24 h, AMs demonstrated different degrees of deformation: rounding and floating, full nuclear condensation and formation of several intracellular vacuoles. Additionally, the number of cellular processes and pseudopods was significantly reduced. The increased number of intracellular particles visibly reduced cellular transparency. The distribution of nanoparticles within and on the surface of cells had an appreciable impact on the growth and metabolism of cells. This resulted in a significantly reduced number of viable cells.
-Cytotoxicity of nanoparticles in AMs: After treating for 24 h, a dose-dependent cell viability reduction could be observed in nanoparticle-treated cells. After exposure to varying doses of the ZnO nanoparticles for 24 h, the lactate dehydrogenase (LDH) levels in the cell media resulted in dose-dependent elevation. Compared with the control, after treatment with 25–100 µg/mL of nano-ZnO, LDH levels in the cell media were greater.
-Effects of nanoparticles on the phagocytosis of AMs: After treatment with varying doses of nano-ZnO for 24 h, AM phagocytosis decreased in an explicit dose-dependent manner. When the concentration was only 10 µg/mL, the treatment with nanoparticle could still reduce the phagocytosis of AMs significantly compared with that observed in control cells. After the exposure dose was increased to 25–100 µg/mL, nano-ZnO caused a significantly greater reduction in AM phagocytosis.

Effect levels

Dose descriptor:
other: Observations
Basis for effect level:
other: see 'Remark'
Remarks on result:
not measured/tested
Remarks:
Effect level not specified (migrated information)

Applicant's summary and conclusion

Conclusions:
In vivo exposure of ZnO nanoparticles to rat caused an increase in oxidative injury to the lungs and disorders in regulating the cytokine network, which were detected in the bronchoalveolar lavage fluid, suggesting that oxidative stress might be important for inducing the respiratory immunotoxicity of nanoparticles. In vitro, the phagocytic function of alveolar macrophages (AMs) was dose-dependently reduced by nanoparticles, which were coincident with the results of multiple measurements, such as a cell viability assay by WST-8 and LDH measurements.
Executive summary:

The study was conducted to investigate the respiratory tract immune toxicity (“immunotoxicity”) of ZnO nanoparticles in vivo and in vitro.

In the in vivo study, 6 rats/group were exposed to nanoparticles by intratracheal instillation at 0, 3.5 and 17.5 mg/kg bw/day once every 2 d for 5 weeks. 24 h after the last exposure, the right lung was then lavaged with normal (physiological) saline to collect bronchoalveolar lavage fluid (BALF) in which both oxidative damage and cytokines were detected. In the in vitro study, alveolar macrophages (AMs) were exposed to the nanoparticles for 24 h and were then analyzed for morphological changes and cell viability.

Exposure to nanoparticles in vivo caused an increase in oxidative injury to the lungs and disorders in regulating the cytokine network, which were detected in the bronchoalveolar lavage fluid, suggesting that oxidative stress might be important for inducing the respiratory immunotoxicity of nanoparticles. In vitro, the phagocytic function and cytotoxicity of AMs was dose-dependently reduced by nanoparticles , which were coincident with the results of multiple measurements, such as a cell viability assay by water-soluble tetrazolium-8 and lactate dehydrogenase measurements.