1. IANH and ISO Proposals C. Michael Garner Garner Nanotechnology Solutions Visiting Scholar Stanford University EE Department

2. 2 Why Established the IANH? Lack of commonly agreed biological testing protocols for nanomaterials Variability of biological response for the “same” nanomaterial in different labs Variability of testing results is slowing progress in scientific understanding of nanotoxicology Multiple groups calling for development of reproducible testing protocols IANH formed to address this “Gap”

4. 4 IANH Goals Establish in vitro and in vivo nanomaterial NanoEHS testing protocols that are validated to produce the same results in multiple laboratories internationally. –Round Robin Testing of Nanomaterials Identify nanoEHS in vitro testing protocols that correlate with results in animal test and may be predictive of in vivo effects. In vitro: Cell level testing In vivo: Animal testing

5. 5 The Challenge Different laboratories testing nano-bio interactions on the same nanomaterial can report very different results. Potential sources of differences –Differences in material preparation –Chemical contaminants –Differences in the biological organisms –Differences in the time required for characterization (leeching of the die from the cells) –Differences in characterization metrology & software

6. 6 IANH Approach Validate Reproducible Methods for –Characterization of material properties –Preparation of nanomaterials –Preparation of biological organisms –Exposure of biological organisms to nanomaterials –Characterization of the nano-bio interactions

7. 7 IANH In Vitro Methodology What Data and Correlations are Needed? 2. 1. Select Cell Line Appropriate to High Risk for Exposure to NP and End Points Validate Ability to Grow Healthy Organisms Validate Ability to Characterize Nanoparticle Properties (Correlated with other labs) Validate properties of Biointeraction Control NanoParticles Characterize Biological Interactions of Nanoparticles with Cells Characterize Biological Interactions Of new particles

8. 8 Progress Groups characterized DLS capabilities –NIST 8012 30nm Au reference material –IRMM 304 60nm silica reference material Groups characterized size (DLS), Zeta potential & pH –160nm CeO 2 –60nm polystyrene Initial in vitro Cell Viability training set on CeO 2 and positive polystyrene Improved in vitro cell viability protocol to remove ambiguities in procedures and used same serum –Repeated cell viability on CeO 2 and positively charged polystyrene

9. Progress Cont. EMPA and NIST developed a tray layout to validate that a lab and the tester can reproduce chemical toxicity with the cell line. Tested the improved protocol and tray layout with a smaller group. –ST-01 (Anatase TiO 2 ) and CuO dispersions 9

10. IANH Status IANH became inactive due to loss of funding and retirement of a coordinator. –IANH was a volunteer effort with only one group having funding to provide biological and nanomaterials A few IANH members became funded by NIEHS –Implemented round robin testing –Made further improvements in IANH Cell Viability Protocol 10

11. 11 General Information Required for an Assay Protocol Cell source & batch –Number of cell generations & passages Serum Batch number Assay Batch Number Cell growth control Cells & assay control Positive Control with Cells Dose Response Nanoparticle Dose Response with Cells Other Assay Specific measurements

12. ISO Proposal Proposed to ISO two new work items –Effect of Nanoparticles on Cell Viability (MTS Assay) –Effect of Nanoparticles on Cell Oxidative Stress [CM- H 2 DCFDA (DCF) Assay] Introduced at ISO International Meeting –Identified existence of ISO Spec 10993-5 [Testing biological compatibility of medical devices (in-vitro)] Working with ANSI to get approval of NWIP 1 & NWIP 2 12

13. Status NWIP 1 & NWIP 2 submitted to ANSI TAG WG 3 –Improvements identified required for approval Improvements have been implemented and the documents put into ISO format –TAG WG 3 will review improvements and make a recommendation –Need to get the supporting data in a presentable format –May need additional experiments Approval required at multiple levels –Determine whether appropriate as a Spec. or other ISO Document 13

14. 14 Characterization for MTS Assay Protocol Source & batch number of cells Serum batch number Number of cell generations & passages Dose response for control (6 & 24 hours) –Cell & Assay control –Cell & Positive control particle –Other controls (Anna.. Dose response for target particles (6 & 24 hour)

15. 15 Dose Response for CM-H2DCFDA Assay (H2O2 Production) Protocol Source & batch number of cells Serum batch number Cell viability control with and without assay (1, 3, 6, 24 hour) Cell dose response to positive controls (1, 3, 6, 24 hour) Cell dose response to target particles (1, 3, 6, 24 hour) Description of cells in the wells compared to the chart (floating, etc.)

16. 16 Validate Healthy Cell Growth Protocol & cell seeding Methodology for Healthy Cell Growth –Number of viable cell passages –Serum assay Pictures of Healthy and Unhealthy Cells Cell Growth Rate Curve Cell viability counts live vs. dead Cell uniformity in the wells

17. Cell Viability 96 Well Layout Determine whether a lab can reproduce chemical toxicity 17 EMPA and NIST