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mechanistic studies
Type of information:
experimental study
Adequacy of study:
supporting study
other: not rated (non-standard test system)

Data source

Reference Type:
Toxicity of silver nanoparticles at the air-liquid interface
Holder, A.L & Marr, L.C.
Bibliographic source:
Bio. Med. Res. Int. 2013, 1-11

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
The objective of this work is to evaluate the toxicity of commercially available aerosolized silver nanoparticles on human alveolar epithelial cells exposed at the air-liquid interface.
GLP compliance:
not specified
Type of method:
in vitro
Endpoint addressed:
acute toxicity: inhalation

Test material

Constituent 1
Reference substance name:
EC Number:
EC Name:
Cas Number:
Test material form:
solid: nanoform
Details on test material:
- Name of test material (as cited in study report): silver (30 - 50 nm coated with polyvinyl pyrrolidone, PVP 0.2% wt) (from commercial supplier: NanoAmor, Houston, TX, USA)
- Physical state: nanomaterials

Test animals

Details on test animals or test system and environmental conditions:
Not applicable - Since this is an in vitro study there is no information on test animals.

Administration / exposure

Route of administration:
other: human alveolar cells are exposed to aerosolized particles
other: carbon dioxide
Details on exposure:
Nanoparticle stock suspensions were prepared by dispersing the particles in sterile nanopure water with a probe sonicator (Misonix 3000) at a concentration of 0.5 mg/mL.
The size distribution in suspension was measured by dynamic light scattering (DLS, Malvern Zetasizer Nano). A drop of the suspension was dried on a transmission electron microscope (TEM) grid, and samples were then analyzed with a TEM (Philips EM420). Elemental analysis was performed with a scanning electron microscope (FEI Quanta 600 FEG) equipped with an energy dispersive X-ray spectrometer (EDX, Bruker Quantax 400).

The exposure chamber consisted of an electrostatic precipitator (ESP) and collagen-coated Transwells (Corning, 12 mm inserts, 0.4 µm pore size, 1.12 cm^2 growth surface), which contained the cells.
The chamber is constructed of two aluminum plates (15.2 cm in diameter, 6.4 cm thick) forming the top and bottom surfaces and an acrylic pipe (14.6 cm in diameter, 3.5 cm in height) forming the cylindrical wall. Four equally spaced inlets around the acrylic cylinder allow four wells to be exposed simultaneously. The inlet air flows over the Transwells and exits through an outlet in the center of the top plate. An electric field is generated in the chamber by connecting the lower plate to a negative high-voltage DC supply (EMCO, model 4120N) and the upper plate to ground.
The Transwells are placed upside down, and cells are grown on what is now the top side of the Teflon membrane (typically the bottom side), in order to minimize the vertical distance that particles must travel before depositing on the cell surface. This orientation maximizes deposition efficiency.
Particle deposition on the Teflon membrane (i.e., the Transwell cell culture surface) was measured with a fluorescein aerosol of a single diameter. The deposited fluorescein particles were collected and fluorescein was extracted and measured on a plate reader (Molecular Devices, SpectraMax M2).
The deposition efficiency was calculated as the percentage of mass depositing on the Transwell relative to the total mass entering the inlet, which was derived from measurements of particle number concentration by a condensation particle counter (CPC, TSI model 3025A). The deposition efficiency for each particle diameter (50, 75, and 100 nm) was measured in three wells in three separate experiments, except for 50 nm, which was measured in four separate experiments. In exposure experiments, the dose of nanoparticles depositing on the cells was calculated by applying the deposition efficiency to the inlet aerosol concentration. Although the nanoparticles tested have higher densities than the fluorescein particles, the deposition efficiencies are not affected.

Aerosols were generated with a constant output atomizer (TSI, model 3076).The nanoparticle aerosols were dried with a diffusion dryer, charge neutralized with a Kr85 source (TSI, model 3012), and mixed with CO2 to a concentration of 5%. The size distribution was measured with a scanning mobility particle sizer consisting of a differential mobility analyzer (TSI, model 3081) and the CPC. Aerosol samples for electron microscopy were collected by placing a TEM grid on a Transwell inside the ESP.

Cells were plated on collagen-coated Transwell inserts at a density of 10^5 #/cm^2 following a protocol modified from Gohla et al. (1996)*. Inserts were turned upside down and 0.15 mL of cell suspension was placed on the bottom of the insert. The insert was placed inside an incubator at 37°C with 5% CO2 for 3 hours while the cells attached to the Teflon membrane. The excess medium was removed, and the inserts were placed with the right side up in a 12-well plate and grown submerged (1.0 mL medium in the bottom chmaber, 0.5 mL medium in the upper chamber) for two days before the exposure.
In preparation for all ALI exposure, the Transwell inserts were placed upside down insdie sterile glass wells (2.6 cm in diameter, 2.2 cm deep), 8 mL of medium was added to the well, and 0.1 mL of medium was placed on top of the inserts to prevent the insert from drying out. The glass wells and inserts were then placed inside the chamber for the duration of the aerosol exposure. A second group of wells was pplaced in an identical chamber to serve as the control group. Each chamber was wiped down with ethanol before the exposure to maintain sterility.
Two ALI exposure scenarios were used in this study: whole aerosol (polydisperse) exposure and single-diameter (monodisperse) exposure. For both exposure scenarios, a control experiment with no particles was conducted simultaneously.
The cells were dosed at the ALI for 2 hoursr with the whole aerosol or 3 hrours with a single-diameter aerosol. After being dosed, the inserts were returned to a 12-well plate, where they were incubated submerged in 1.0 mL of F12K media with 10% FBS at 37∘C with 5% CO2 for 24 hr. The media was
then collected to measure LDH and IL-8 concentrations, and the MTT assay was begun. Each ALI exposure condition was done once on triplicate wells.

Cells were exposed to nanoparticles in liquid suspensions. Cells were plated in 12-well plates at a density of 10^5 #/cm^2 and grown for two days before an exposure. Cells were dosed with the nanoparticle suspension (1 mL per well; dose levels: 2.6, 6.6 abd 13.2 µg/cm^2) and kept in an incubator at 37°C with 5% CO2 for 24 hours. A single dose (rather than repeated dosing) and the 24 hr incubation period were selected. After the exposure, the medium was collected for the LDH assay and for IL-8 measurement, and the MTT assay was begun. Each suspension exposure condition was done once on triplicate wells.

- A. Gohla, K. Eckert, and H. R. Maurer, “A rapid and sensitive fluorometric screening assay using YO-PRO-1 to quantify tumour cell invasion through Matrigel," Clinical and Experimental Metastasis, vol. 14, no. 5, pp. 451 - 458, 1996.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Please refer to "Details on exposure"
Duration of treatment / exposure:
Air-liquid interface exposure: 2 hours (whole aerosol) and 3 hours (single diameter aerosol)
Suspension exposure: 24 hours
Frequency of treatment:
Doses / concentrationsopen allclose all
Doses / Concentrations:
0.7 µg/cm^2
other: dose for air-liquid interface exposure
Doses / Concentrations:
2.6, 6.6 and 13.2 µg/cm^2
other: estimated dose for suspension exposure
No. of animals per sex per dose:
not applicable
Details on study design:
All experiments were performed with a human alveolar cell line (A549, Sigma ECACC). Cells were grown in F12 medium using Kaighn's modification (F12K, Invitrogen) with 10% fetal bovine serum (FBS, invitrogen) and 1% antibiotic/antimycotic (Invitrogen).


Measurements of cellular response were made with several assays (Carlson et al., 2008, Lu et al., 2009, Ayres et al., 2008)*. Cytotoxicity was assessed with a lactate dehydrogenase (LDH) assay (kit from Sigma) and Methylthiazole tetrazolium (MTT) assay (Sigma). The exposure medium was collected after nanoparticle exposure and centrifuged to remove the nanoparticles from the medium. The extracellular LDH concentration in the supernatant was measured. The metabolic activity of the cells ws measured with an MTT assay. A proinflammatory response was assessed by measuring the secretion of the pro-inflammatory cytokine interleukin 8 (IL-8) with an ELISA assay (kit from Invitrogen). The exposure medium was collected and centrifuged to remove the nanoparticles. The supernatant was kept frozen at -8°C until the assay was performed.

- C. Carlson, S. M. Hussein, A. M. Schrand et al., “Unique cellular interaction of silver nanoparticles: size-dependent generation of reactive oxygen species,” Journal of Physical Chemistry B, vol. 112, no. 43, pp. 13608–13619, 2008.
- S. Lu, R. Duffin, C. Poland et al., “Efficacy of simple shortterm in vitro assays for predicting the potential of metal oxide nanoparticles to cause pulmonary inflammation," Environmental Health Perspecctives, vol. 117, no. 2, pp. 241-247, 2009.
- J. G. Ayres, P. Borm, F. R. Cassee et al., “Evaluating the toxicity of airborne particulate matter and nanoparticles by measuring oxidative stress potential—a workshop report and consensus statement,” Inhalation Toxicology, vol. 20, no. 1, pp. 75–99, 2008.
Positive control:
Nickel oxide nanoparticles were used as positive control as they have previously been shown to generate more reactive oxygen species compared to other nanoparticles and cause a cytotoxic response in the A5449 cell line (Lu et al., 2009)*. The dose used was 2.1 µg/cm^2 at the air-liquid interface.

- S. Lu, R. Duffin, C. Poland et al., “Efficacy of simple shortterm in vitro assays for predicting the potential of metal oxide nanoparticles to cause pulmonary inflammation," Environmental Health Perspectives, vol. 117, no. 2, pp. 241 - 247, 2009.

Results and discussion

Details on results:
Fluorescein particle deposition on the Teflon membrane was measured and were as follows (deposition efficiency):
50 nm (diameter): median 38.2%; 25th: 32.5%; 75th: 63.1%
75 nm (diameter): median 63.3%; 25th: 53.1%; 75th: 74.9%
100 nm (diameter): median 63.5%; 25th: 52.7%; 75th: 75.5%

Atomizing the suspension of silver nanoparticles resulted in an aerosol that consisted of particles with a geometric mean diameter of 37 nm and a volume weighted geometric mean diameter of 169 nm. Electron microscopy confirmed that the aerosol particles had the same physical characterisitcs as the silver nanoparticles in suspension. The particles were approximately spherical with diameters of ~50 nm and were composed of silver with a crystalline diffraction pattern.

Both suspensions and aerosolized nanoparticles caused negligible cytotoxicity and only a mild inflammatory response.

Applicant's summary and conclusion

According to the authors, both suspensions and aerosolized nanoparticles caused negligible cytotoxicity and only a mild inflammatory response.