CEINT/ NIST Protocol for Preparation of Nanoscale TiO2 Dispersions in Biological Test Media for Toxicological Assessment, Version 1.1

Toxicity and fate assessment are key elements in the evaluation of the environmental, health and safety risks of engineered nanomaterials (ENMs). While significant effort and resources have been devoted to the toxicological evaluation of many ENMs, including nanoscale TiO2, obtaining conclusive and reproducible results continues to be a challenge. This can be traced in part to the lack of standardized dispersion protocols and the inconsistent application of dispersion procedures in relevant biological and environmental matrices. In order to address these issues, the National Institute of Standards and Technology (NIST) jointly with the Center for the Environmental Implications of Nanotechnology (CEINT) have developed a series of standardized and validated protocols for the dispersion of ENMs from a powdered material source for both human health and environmental testing applications. These protocols have been developed and validated using NIST Standard Reference Material (SRM) 1898[1]. SRM 1898 consists of a widely studied and industrially relevant TiO2 nanomaterial with broad commercial penetration and a production history dating back several decades

While the procedures detailed in this document focus on the dispersion of SRM 1898 in specific aqueous media, it is believed that the adopted characterization, optimization and validation approaches can be more generally applied to the preparation of ENM dispersions in any relevant matrix. For this reason, and to allow for a broader applicability of this work, experimental details and discussion regarding the characterization, process optimization and validation adopted for the development of the dispersion method will be detailed and discussed in a separate and forthcoming publication.

In this protocol, TiO2 nanoparticle dispersions in relevant biological media are produced by following a series of steps applied to a stock TiO2 aqueous nanoparticle dispersion. To prepare the stock TiO2 aqueous dispersion, refer to the previous protocol in this series.

The protocol focuses on two representative media broadly used for in vitro assays - phosphate buffered saline solution (PBS) and Dulbecco's Modified Eagle Medium containing 10 % fetal bovine serum (DMEM-FBS). In both cases, bovine serum albumin (BSA) is utilized as a stabilizing agent; series specific serum albumin has been demonstrated to function as a non-specific biocompatible stabilizer. This protocol is proposed for the preparation of dispersions for generic acute bioassay applications; its use for chronic toxicity evaluations is beyond the scope of this work and should be validated by use of proper controls.

The method described herein, if applied correctly, yields 100 mg/mL monomodal nanoscale TiO2 dispersions in PBS or DMEM-FBS, characterized by mean particle diameters of ≈ 75 nm (in PBS) and ≈ 83 nm (in DMEM-FBS) and pH values from 7.2 to 7.4 (in PBS) and ≈ 7.8 (in DMEM-FBS). The dispersions retain their particle size distribution for 48 h, under relevant incubation conditions (in DMEM-FBS) and at room temperature (in PBS). Dispersions prepared following this protocol should be stored so as to minimize exposure to light (e.g., in amber vials).

[1] SRM 1898 was in production as of the publication of this protocol, with a target date for release in Fall 2011. The NIST SRM inventory can be accessed at http://www.nist.gov/srm/.

DOI: http://dx.doi.org/10.6028/NIST.SP.1200-4