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July 2006 Symposium on Nanotechnology and the Environment: Potential Exposure Scenarios and Potential Toxicity of Nanomaterials: Highlights, Question and Answer Session Session

July 12, 1:00-2:00 pm

Dr. David B. Warheit, DuPont Haskell Laboratory for Health and Environmental Sciences.


Dr. Warheit’s research involves health effects resulting from respiratory exposures to nanomaterials. There are parallel research tracks: generic mechanistic research, and product-specific testing.

Nanoparticles are equivalent in size to ultrafine particles (<100 nm). Ultrafine nanoparticles are <100 nm in size, and fine particles are 100 nm to 3 µm in size. Certain particle sizes are respirable (e.g., <3 µm in rat, <5 µm in human).

Regarding lung structure as it relates to particle deposition: rats have 3 to 5 bronchoalveolar duct bifurcations; humans may have 15 to 20. Ciliated Clara cells are positioned at the junctions of bronchial tubes and alveolar duct bifurcations. At the first of these junctions is where inhaled particles deposit in the distal lung. Macrophages engulf foreign particles to clear them from the lung. They engulf the particles, then move to the mucociliary escalator on the airway surface, and move up the bronchial tubes to be coughed up or swallowed.

Some common perceptions of pulmonary toxicity include the idea that nanoparticles are more inflammogenic and/or tumorigenic than fine-sized particles of identical chemical composition. However, not all nano-sized particles are more toxic. Some factors that may influence toxicity are surface coatings, species differences, particle aggregation potential, and whether the particle was fumed vs. precipitated in its manufacture.

Pulmonary bioassays can be used as measures of lung toxicity. It is important to consider dose response characteristics and the time post-exposure. Data from 24 hours post-exposure are not as useful because there is always an inflammatory response from the intratracheal instillation exposure. Thus the sustainability or nonsustainability of the response is very important (often measured at 1 week, 1 month or 3 months postexposure).

There can be a heterogeneous cellular response in the lung to inhalation of crystalline silica – macrophages, neutrophils and lymphocytes can respond. This can be indicative of an ongoing inflammatory response. Alternatively, the cellular response can be homogeneous (macrophages only). This is indicative of no sustained inflammation.

Regarding lung toxicity, generally there has been good correlation between the findings of instillation and inhalation studies.

In an experiment with TiO2, two concentrations of nano sized particles (nanoscale rods or nanoscale dots) were tested and compared with fine sized TiO2 particles. The positive control was quartz particles (Min-U-Sil). Physical-chemical characterization of the particles was robust -- crystal form, crystal size, shape, surface area etc. were measured and recorded. The surface area of Nanorods was four times greater than the corresponding fine particles, and the surface area of Nanodots was thirty times greater than fine TiO2 particles. Following intratracheal instillation of particles into the lungs of rats, there were no toxicity differences among the groups of TiO2 – exposed rats for various biomarkers. The quartz positive control produced an active inflammatory response.

Nanoquartz is a form of alpha quartz crystalline silica. The hypothesis states that nanoquartz should be more toxic than fine-sized Min-U-Sil quartz particles, but results show that one nanoquartz sample was less toxic and another one was equivalent in toxicity to Min-U-Sil. Three quartz-particle types of different sizes were tested along with a negative control. Nanoquartz 2 (size: 12 nm) produced a greater or equivalent response compared to Min-U-Sil, and fine quartz was less toxic than Min-U-Sil. Nanoquartz 2 has eighteen times the surface area of Min-U-Sil. All quartz particle-types produced ongoing inflammatory responses to varying degrees. Min-U-Sil and Nanoquartz 2 produced greater accumulation of macrophages that did not get cleared.

A red blood cell hemolysis assay can be useful as a measure of surface reactivity for quartz samples. In our study, surface reactivity was more predictive for toxicity than surface area or size. Hemolytic potential does not correlate well for other particle types.

Inhalation of zinc oxide particles can cause metal fume fever. Tests were performed by Dr. Warheit to see if nano-sized zinc oxide was more potent than fine zinc oxide. As an aerosol, the particles were observed to aggregate in the inhalation chamber. The question then arises, following deposition in the lung - do particles then behave as an aggregate or do they disaggregate? Both nano-sized and fine zinc oxide produced aggregates of similar sizes and both had similar toxicity profiles based on pulmonary biomarkers.

To investigate impacts of particle surface coatings, six grades of TiO2 with various coatings/surface treatments were examined. Although titanium dioxide is generally considered a low toxicity material, the TiO2 with the highest level of surface coating caused an inflammatory response that was maintained longer than for the other TiO2 samples, and also produced the greatest cytotoxicity (still minor compared to quartz particles).

To summarize, it cannot be assumed that nanomaterials are the same as their bulk counterparts. The chemistry and physics (physical properties) change as one moves down the nanoscale – but what about the biology or toxicology? Each particle type should be tested on a case-by-case basis.

Question-and-Answer Session

A questioner asked what causes the toxicity of nanoquartz. Dr. Warheit answered that quartz is listed as a Class 1 human carcinogen; it is a variable entity. Epidemiologists believe that quartz has lead to lung cancer and fibrosis in some cases but not in others; experts don’t know why. There may be a difference between synthetic and mined quartz; iron content may play a role. A questioner asked whether one can correlate inflammation and cytotoxicity with the amount of material removed via the mucociliary elevator. Dr. Warheit indicated that it depends on particle type. Those not cleared can cause inflammation. Even low toxicity materials have clearance half-times of 55 days in rats and much longer in humans. Overloading can produce longer clearance times, and inflammation. A commenter stated that the standard for “nano-sized” materials had been set at approximately 1-100 nanometers and that he found it curious that hemolysis was used instead of a cell apoptosis test. The commenter wondered if Min-U-Sil could be used as a positive control for hemolysis. Dr. Warheit responded that hemolysis studies were performed using fourteen particle types. The preliminary conclusion was that it was not instructive for particles other than quartz and that most available nanoparticle studies are in vitro. Comparisons have been done in vitro and in vivo. For diesel extracts, in vitro tests have given opposite results compared to the in vivo tests when assessing toxicity ranking of the same particulate materials. In a current ongoing study comparing in vitro results with in vivo effects for 5 different dusts, the in vitro data appear to be problematic in terms of validating the in vivo results.

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