Comment submitted by Samantha Dozier, Nanotechnology Policy Advisor, Regulatory Testing Division, People for the Ethical Treatment of Animals (PETA)

Samantha Dozier, Ph.D. Nanotechnology Policy Advisor Regulatory Testing Division People for the Ethical Treatment of Animals 501 Front St. Norfolk, VA 23510 Document Control Office (7407M) Office of Pollution Prevention and Toxics Environmental Protection Agency 1200 Pennsylvania Ave., NW Washington DC 20460-0001 Attention: Docket ID Number EPA-HQ-OPPT-2004-0122 Risk Management Practices for Nanoscale Materials The EPA is considering a stewardship program for nanoscale materials under the Toxic Substances Control Act (TSCA) and is therefore requesting information to address specific areas of risk management for nanomaterials. These areas include: (1) risk management practices currently used for nanoscale materials; (2) risk management practices that could potentially be used for nanoscale materials; (3) rationale for the use of these practices and the effectiveness/ efficacy of these practices; (4) and issues to consider for determining risk management practices for nanoscale materials to include in the basic program. In addition, EPA is interested in gathering information on any other risk management practices for nanoscale materials that should be considered for the voluntary program or the in-depth program. Nanotechnology is the convergence of the most modern, high-tech sciences that exist today and can be defined as the purposeful design, characterization, production, and application of structures, devices, and systems by controlling shape and size at the nanometer scale. Nanotechnologies are integral to fields as varied as computer science, cosmetics, and medicine. However, reaching an agreed upon definition for the field of nanotechnologies has proven difficult. Unifying characteristics that help define nanoparticles include: 1. The size of nanochemicals and nanomaterial end-products fall between 1 and 100 nanometers in length in at least one direction; 2. Nanoparticles demonstrate size-dependent physicochemical properties; 3. Nanochemicals have unique surface structure compared to their micro sized counterparts; 4. Their solubility and aggregation patterns are dependent on the surface-to-area ratio. Physicochemical properties of nano-sized chemicals differ from their micro-sized counterparts of the same composition. These differences are accounted for by the increased surface area of the nanochemicals with respect to traditional chemicals and the concomitant increase in the number of particles that humans may be exposed to per unit of mass [1, 2]. Determining Physicochemical Attributes of Nanoparticles Because nanomaterial production is not yet standardized, the resulting nanomaterials are non-uniform and are riddled with various heavy metal contaminants. Batch-to-batch variation in nanomaterials produced within the same manufacturing facility, as well as variability found in the same nanochemical made by different manufacturers, requires the use of high throughput, modern methods of synthesis and analysis so that researchers can know the exact composition of the nanomaterial with which they are working to improve and insure consitent quality of product (Finnish Presidency Conference ? Safety for Success). In addition, experiments aimed at elucidating the toxicity of these compounds using non-standardized methods results in a hodge-podge of uninterpretable toxicity data. These problems are recognized by researchers and regulatory agencies and have proven insurmountable by traditional low-throughput, expensive, and irreproducible animal-based toxicology methods. Standardized methods that allow manufacturers and researchers to know the exact composition and level of purity of the nanomaterial they are studying are paramount to attaining useful toxicity data down the road [1, 3, 4]. The best methods available to measure and test the purity of nanomaterials include: scanning electron microscopy (SEM) with energy dispersive X-ray (EDX) analysis which measures nanomaterialsize and detects dispersion, non-carbon-based contamination, and geometry of the nanomaterial; transmission electron microscopy (TEM) answers questions pertaining to morphology and purity of the nanomaterial?s surface; and thermogravimetric analysis (TGA) that analyses a wide range of parameters, including compositional analysis, decomposition temperature, rate of decomposition, a quantitative measure of mass change associated with transition and thermal degradation and nanomaterial oxidation. Using the instruments described above will allow basic, standardized measures of nanomaterials and would encourage manufacturers and researchers to move towards a standard definition of each nanomaterial. The ability to describe physicochemical properties in a consistent manner would also better insure the safety of workers and researchers because heavy metal contaminants would be well defined and appropriate safety measures could be employed. Instruments such as scanning mobility particle sizers (SMPS) that can measure the size of particles between 3 and 1000 nanometers (nm) and scanning electron microscopes (SEM) that utilize an electron dispersive spectrometer (EDS) make nanoparticles countable and their chemical compositions discernable. A recently announced advance in nanoparticle detection for workplace safety comes from Dekati Ltd. with the advent of the Electrical Dekati Industrial Hygiene Particle Sensor (EDiPS) (Finnish Presidency Conference ? Safety for Success). This sensor is portable and offers real-time nanoparticle measurements to insure workplace safety. Dekati Ltd. expects the EDiPS to be available commercially by the end of this year. Nanotoxicity Testing: an Opportunity to Employ Modern Methods Because nanotechnology incorporates many scientific fields while at the same belonging to a unique discipline, debate has erupted about the suitability of applying decades-old methods to this new field. In addition to the ethical problems of animal experimentation, there are clear and indisputable scientific limitations for animal experiments, especially for the field of nanotechnology [1, 5-8]. For the past twenty-five years, the field of drug discovery has encountered pitfalls and dead-ends directly linked to animal experimentation. This ineffective approach, which has resulted in an epidemic waste of money and lives, cannot be tolerated for the burgeoning field of nanotechnology. A wealth of scientific advances during the last two decades have led to the development of several efficient and reliable non-animal drug and toxicity testing protocols. Human-relevant methods are available at this time and should be the basis of the in vitro nanomaterial-testing paradigm that the field direly needs. For example, several reproducible, reliable and human-relevant in vitro assays are based on various applications of human cell lines. In the broadest sense, these assays have been in use for almost two decades; however, within the last decade cell culture has proven to be an excellent method for toxicity testing. There are many research groups working to tailor cell culture protocols to the field of nanotechnology. A group with recent success is that led by Barbara Panessa-Warren of the Department of Energy?s Brookhaven National Laboratory; this group published a method of cell culture expressly designed for screening nanomaterials [9]. Panessa-Warren?s group visualized the human cells with sophisticated imaging methods that allow researchers to follow the cell-nanomaterial interaction ultrasonically throughout the entire experiment. The group tested both lung and colon cells ? which depict typical routes of exposure ? to assess the toxicities of various carbon-based nanomaterials. Their findings show that carbon nanotubes disrupted cell-cell adhesions and morphology, caused a change in the growth profile and induced apoptosis, and that these changes were related to time of exposure rather than exact dose. This novel experimental advance is a significant step towards nanomaterial-specific toxicity testing standards and is the type of science needed for the field. Many groups have used human cell culture in concert with microarray experiments and cytotoxicity analyses, which allow detection of early signs of cellular toxicity. Known stress responses can be measured before and after exposure to nanomaterials thereby giving scientists clear indications of cellular responses and avoiding the gruesome endpoints exemplified by animal experimentation [5, 10-14]. An agreed upon set of factors that are known to influence, if not largely determine, a material?s toxicity to human cells and systems are the events that lead to the generation of reactive oxygen species (ROS) in vivo. In Nel et al. 2006, ?Toxic Potential of Materials at the Nanolevel,? a series of established in vitro assessments of ROS activity is proposed as a paradigm for nanomaterials toxicity testing [1]. The author, Andre Nel PhD, is a well-respected nanomaterials expert. He summarizes the most important aspects of toxicity testing by explaining that generation of ROS is among the most predictive of tests that can be done. These assays assess injury to proteins, DNA, and cellular membranes due to oxidative stress. Oxidative stress can be measured by mitochondrial perturbation, specifically inner membrane damage, permeability transition, energy failure, and apoptosis. Table 2 of this review lists thirteen cellular responses to toxic chemicals and the corresponding assays by which these effects can be measured. In addition, Nel specifies that the ultimate goal of the predictive approach to toxicity testing ?would be to develop a series of toxicity assays that can limit the demand for in vivo studies, both from a cost perspective as well as an animal use perspective.? This notable scientist seems to recognize that animal experimentation has severe limitations and is problematic in this modern era [1, 3]. Studies from Dr. Vicki Colvin?s lab, utilize a series of in vitro human cell culture assays predictive of cellular responses to toxic chemicals. In a study entitled ?Nano-C60 cytotoxicity is due to lipid peroxidation,? experiments were performed to assess cytotoxicity/cell viability, lactate dehydrogenate release, mitochondrial activity, DNA content, plasma membrane permeability, lipid peroxidation, glutathione production, and the ability to prevent oxidative damage by the addition of the addition of L-ascorbic acid. By changing the number of hydroxyl groups on the fullerene surface resulted in a reduction of toxicity by several orders of magnitude. These experiments show that fullerene toxicity can be rigorously tested by means of cost-effective, predictive, and relevant in vitro assays. In addition, potential toxicity of the fullerenes was lowered significantly by using these in vitro assays to target chemical aspects of the nanomaterials that contribute to toxicity. The author states that, ?in vitro testing provides a cost-effective means for such studies, and as this report illustrates, cell culture experiments are well suited for developing mechanistic models to inform material development.? In addition, the author explains that this study seeks ?to set a standard for future efforts to characterize the environmental and health impacts of other classes of engineered nanoparticles.? The above studies clearly show that the most efficient (and humane) means of toxicity testing lie in modern, high-throughput in vitro assays [[1, 3, 4, 8]. To assess effects on particular organs, a novel in vitro methodology has been developed, based on a microchip seeded with various cell types and nourished using microfluidics. One such device, the HuREL, allows the scientist to test a compound within a matrix of different cell types, linked by microfluidic channels, can answer questions regarding how nanomaterials interact with and are metabolized by human tissues. Details of this work can be read in depth in Sin et al. 2004, entitled The Design and Fabrication of Three-Chamber Microscale Cell Analog Devices with Integrated Dissolved Oxygen Sensors. Systems such as these will allow scientists to test whether a nanomaterial is effectively targeted to a particular organ or cell, and whether it has detrimental effects on organs such as the kidney, liver, or heart. This system was unveiled last year and has been exciting for both researchers and investors alike[15]. Using this novel technology will save not only human and animal lives, but also time, money, and resources. EPA can best protect humans and the environment by requiring corporations, academic researchers, and laboratories funded by its own Agency to use only the most reliable, high-throughput methods available and to build a foundation of human-relevant testing methods for the field of nanotechnology from its outset. References: 1. Nel, A., et al., Toxic potential of materials at the nanolevel. Science, 2006. 311(5761): p. 622-7. 2. Donaldson, K., et al., Nanotoxicology. Occup Environ Med, 2004. 61(9): p. 727-8. 3. Stone, V.a.D., K., Signs of Stress. Nature Nanotechnology, 2006. 1(1): p. 23-24. 4. Sayes, C.M., et al., Nano-C60 cytotoxicity is due to lipid peroxidation. Biomaterials, 2005. 26(36): p. 7587-95. 5. Ma, S.H., et al., An endothelial and astrocyte co-culture model of the blood-brain barrier utilizing an ultra-thin, nanofabricated silicon nitride membrane. Lab Chip, 2005. 5(1): p. 74-85. 6. Wittig, J.H., Jr., A.F. Ryan, and P.M. Asbeck, A reusable microfluidic plate with alternate-choice architecture for assessing growth preference in tissue culture. J Neurosci Methods, 2005. 144(1): p. 79-89. 7. Zucco, F., et al., Toxicology investigations with cell culture systems: 20 years after. Toxicol In Vitro, 2004. 18(2): p. 153-63. 8. Sayes, C.M., et al., Correlating Nanoscale Titania Structure with Toxicity: A Cytotoxicity and Inflammatory Response Study with Human Dermal Fibroblasts and Human Lung Epithelial Cells. Toxicol Sci, 2006. 9. Panessa-Warren, B., Warren, J., Wong, S., Misewich, J., Biological cellular response to carbon nanoparticle toxicity. J. Phys.: Condens. Matter, 2006. 18(33): p. S2185-S2201. 10. Jia, G., et al., Cytotoxicity of carbon nanomaterials: single-wall nanotube, multi-wall nanotube, and fullerene. Environ Sci Technol, 2005. 39(5): p. 1378-83. 11. Veeriah, S., et al., Apple flavonoids inhibit growth of HT29 human colon cancer cells and modulate expression of genes involved in the biotransformation of xenobiotics. Mol Carcinog, 2006. 45(3): p. 164-74. 12. Magrez, A., et al., Cellular toxicity of carbon-based nanomaterials. Nano Lett, 2006. 6(6): p. 1121-5. 13. Lesniak, W., et al., Silver/dendrimer nanocomposites as biomarkers: fabrication, characterization, in vitro toxicity, and intracellular detection. Nano Lett, 2005. 5(11): p. 2123-30. 14. Yin, H., H.P. Too, and G.M. Chow, The effects of particle size and surface coating on the cytotoxicity of nickel ferrite. Biomaterials, 2005. 26(29): p. 5818-26. 15. Sin, A., et al., The design and fabrication of three-chamber microscale cell culture analog devices with integrated dissolved oxygen sensors. Biotechnol Prog, 2004. 20(1): p. 338-45.