1. A PARADIGM ADVANCING TOXICITY TESTING OF NANOMATERIALS IN THE 21 st CENTURY AND QSAR MODELS DEVELOPMENT. David Y. Lai. U.S. EPA, Washington, DC. Feb. 9, 2012 NCI caBIG ® Nanotechnology Working Group

2. Complexity of Nanomaterials (NM) NM are of varying : Chemical composition Size/Dimension Agglomeration/Aggregation state Shape Surface area Surface chemistry/reactivity Crystallinity Other physicochemical properties

3. Shape Agglomerate/ Aggregate Size Surface area Surface energy Deposition & Clearance kinetics Crystallinity Chemical composition Surface reactivity Biological Effects (e.g., cytotoxicity, inflammation, cancer) Biopersistence Surface chemistry/ charge Solubility & Durability Engineered Nanomateria Physical factors Biopersistence Chemical factors Surface coating Chemical/ metal ions

4. Issues on Chronic Inhalation Toxicity Study Very costly Use large number of animals Take long time to complete Large quantity of materials to generate aerosol Require sophisticated exposure facilities, and are technically difficult

5. In vitro Assays Advantages: Rapid and inexpensive Allow specific biological and mechanistic pathways to be tested under controlled conditions No animal studies needed if an in vitro assay/a battery of in vitro assays can be used for hazard identification

6. Issues on In Vitro Assays of NM Adsorption of proteins: may confound endpoints that rely on the measurement of a protein (e.g., LDH, formazan) produced but removed from the supernatant -- false negative Interference of assays rely on measurement of a colored or fluorescent product –false positive & false negative Culture medium effects: on physicochemical characteristics and particokinetics – false positive and false negative

7. HTS Assays Proposed by NRC for toxicity testing of chemicals in the 21 st Century A powerful tool for monitoring the pattern of cellular pertubations in specific pathways – shifts in gene expression: providing “genetic fingerprints” elicited in vitro and in vivo Can assess the biological activity of larger numbers of NM with different pc properties by multiple cell-based assays, in multiple cell types, and at multiple doses Toxicogenomics and other technologies (e.g., proteomics) – the only possible solution to deal effectively with issues involved in safety assessment of diverse types of NM

8. A Paradigm advancing “Toxicity Testing of Nanomaterials (NM) in the 21 st Century” In this proposed paradigm, the health hazards and molecular mechanisms of various classes/subclasses of NM are evaluated by using reference NM, short-term in vivo animal studies in conjunction with high-throughput screenings and mechanism-based short-term in vitro assays. The toxicological properties of a small number of well characterized reference materials of each class/subclass of NM are first characterized by short-term in vivo studies (e.g., 28- or 90-day inhalation) in rodents. In vivo and in vitro high-throughput genomics and/or proteomics studies are then performed to investigate the underlying molecular mechanisms/toxicity pathways and biomarkers of the toxic responses. Mechanism-based short-term in vitro assays in appropriate cell lines are also conducted to aid in elucidation or interpretation of mechanisms, toxicity pathways and biomarkers data derived from the in vivo studies.

9. A Paradigm advancing “Toxicity Testing of Nanomaterials (NM) in the 21 st Century” (cont.,) Once these mechanistic data on reference NM are obtained, they can be used to benchmark and predict the effects and hazard potential of a particular NM belonging to the same class/subclass by comparing data of their in vitro high-throughput and mechanism-based short-term in vitro assays. With well-designed experiments, testing NM of varying/selected physicochemical parameters may be able to identify the physicochemical parameters contributing to toxicity. When the physicochemical parameter(s) and the cut-off values of the parameter(s) contributing to toxicity are identified, knowledge rule-based or other SAR computer model systems can be developed for predicting the hazard potential of NM based on property-activity relationships.

10. Computerized predictive models development Carbon-based Metal-basedOthers Fullerenes Carbon nanotubes Others (e.g., carbon black ) Metals (e.g.,Ag, Au, Cu, QD) Metal oxides (e.g.,TiO 2, ZnO, CeO 2 ) Dendrimers, nanoclays, polystyrene, etc. Well-characterized reference materials Nanoparticles with varying/ selected physicochemical characteristics Short-term in vivo studies and high- throughput genomics/ proteomics in vivo assays High- throughput genomics/proteom ics in vitro assays Mechanism- based short- term in vitro assays Characterize toxicological effects, identify key toxicity pathways/ biomarkers and molecular mechanisms underlying the effects of reference materials with high and low toxicity Evaluate toxicity/hazard potential by comparing high-throughput and short- term in vitro assays data with those of reference materials Identify physicochemical parameter(s) and cut-off values of parameter(s) that are contributing to toxicity PBPK and dose- response modelings for quantitative risk assessment Hazard ranking and prioritizing for further testing

11. An Example -- CNT (1)Characterize the toxicological effects of well characterized reference nanomaterials (e.g., multi-wall carbon nanotubes, MWCNT) by short-term in vivo animal study (e.g., 28- or 90-day inhalation toxicity studies in rats) Purified MWCNT (Diameter : 1 - 30 nm; Length : > 10 µm) Purified MWCNT (Diameter : 30 - 100 nm; Length : < 5 µm) Purified MWCNT (Diameter : 30 -500 nm; Length : >10 µm)

12. (2) Investigate the molecular mechanisms underlying the effects and identify key toxicity pathway/biomarkers by high- throughput assays. In vivo and in vitro high-throughput genomics and/or proteomics studies of alveolar macrophages, epithelial and mesothelioma cells exposed to MWCNT are performed to investigate the underlying molecular mechanisms/toxicity pathways and biomarkers of the toxic responses.

13. (3) Test the reference nanomaterials (MWCNT) by mechanism-based short-term in vitro assays. Mechanism-based short-term in vitro assays (DCFDA assays for oxidative stress/ROS, ELIZA assay for cytokines, comet assays for DNA damage, TUNEL assays for apoptosis, etc.) in human/rat alveolar macrophages, epithelial and mesothelioma cells are conducted to aid in elucidation or interpretation of mechanisms, toxicity pathways and biomarkers data derived from the high-throughput genomic/proteomics studies.

14. (4) Evaluate the potential hazard/risk of a particular MWCNT by conducting high-throughput in vitro assays and mechanism-based short-term in vitro assays (in human/rat alveolar macrophages, epithelial and mesothelioma cells) and comparing data with those of the three reference materials (MWCNTs). (5) Evaluate the potential hazard/risk of MWCNT of varying physicochemical parameters (e.g. diameter, length, aspect ratio, agglomeration state) by conducting high- throughput in vitro assays and mechanism-based short- term in vitro assays and comparing data with those of the three reference materials (MWCNTs).

15. (6) Identify physicochemical parameter(s) and cut-off values of parameter(s) that contribute to toxicity of MWCNT. (7) Develop computer models to predict toxicity of MWCNT based on property-activity relationships. Once the physicochemical parameter(s) and the cut-off values of the parameter(s) [e.g. length, diameter, agglomeration state, density] that contribute to toxicity are identified, knowledge rule-based or other SAR computer model systems can be developed to predict the hazard potential of particular MWCNT.

16. References Gou NA, Onnis-Hayden A, Gu AZ. (2010). Mechanistic toxicity assessment of nanomaterials by whole-cell-array stress genes expression analysis. Environ Sci Tech, 44:5964-5970. Haniu H, Matsuda Y, Takeuchi K, Kim YA, Hayashi T, Endo M. (2010). Proteomic-based safety evaluation of multi-walled carbon nanotubes. Toxicol Appl Pharmacol, 242: 256-262. Lai D.Y., Sayre P.G. (2009). Toxicity Testing and Evaluation of Nanoparticles: Challenges in Risk Assessment. In: Nanotoxicology From In Vivo and In Vitro Models to Health Risks. (SC Sahu, DA Casciano, Eds.), Chapter 21, Wiley. Lai, D.Y.(2011). Toward Toxicity Testing of Nanomaterials in the 21 st Century: A Paradigm for Moving Forward. WIREs Nanomed Nanobiotechnol 2011. doi:10.1002/wnan.162 Poma A, DiGiorgi ML, (2008). Toxicogenomics to improve comprehension of the mechanisms underlying responses of in vitro and in vivo systems to nanomaterials: A review. Current Genomics, 9:571-585. Rollo R, France B, Liu R, et al., (2011). Self-organizing map analysis of toxicity-related cell signaling pathways for metal and metal oxide nanoparticles. Environ Sci Tech. 45:1695-1702. Witzmann FA, Monteiro-Riviere NA. (2006). Multi-walled carbon nanotube exposure alters protein expression in human kerotinocytes. Nanomedicine, 2:158-168.