Challenges for Evaluation of the Safety of Engineered Nanomaterials

by Linda J. Johnston, Norma Gonzalez- Rojano, Kevin J. Wilkinson, and Baoshan Xing

Nanotechnology has developed rapidly in the last two decades with significant effort focused on the development of nano-enabled materials with new or improved properties that offer solutions for current world challenges. The commercialization of products containing engineered nanomaterials (ENM) has progressed much more rapidly than the development of practical approaches to ensure their safe and sustainable use. The lack of adequate detection and characterization techniques and reproducible and validated methods for toxicological studies have been identified as major limitations. The rapid development of ENM of increasing complexity and diversity and concerns over the adequacy of existing regulations also contribute to safety concerns with these materials. The full potential of nanotechnology can only be realized when feasible, cost-effective strategies to ensure a safe-by-design approach, effective risk assessment approaches and appropriate regulatory guidelines are in place.

An IUPAC-sponsored Workshop on the Safety of Engineered Nanomaterials in Queretaro, Mexico in late 2017 aimed to foster a greater awareness of the challenges and identify future research needs that must be filled to develop a regulatory framework for nanomaterials. The workshop covered four topics: (1) Detection and characterization of ENM, (2) Transformation of ENM in consumer products and the environment, (3) Nanotoxicology methods and gaps for environmental health and safety (EHS) and (4) Challenges for metrology, risk assessment, and standardization. This article highlights the main challenges and potential solutions that were developed during the workshop (https://iupac.org/project/2016-045-2-700) and subsequently by the authors and published in a recent NanoImpact article [1].

Detection and characterization of ENM

The utility of many published studies on the synthesis, applications, and toxicology of ENM is limited by inadequate characterization and data reporting. These issues impact quality control during production of the materials and prevent inter-laboratory comparability for applications development and nanotoxicology studies. Several factors contribute to this problem.

First, the characterization of ENM is complex, compared to conventional chemicals, since many properties must be assessed (Figure 1) and it is not always clear which ones are important for a specific application. ENM properties can be separated into two categories: intrinsic properties such as size, surface area, and composition that do not depend on the medium or environment (“what they are”) and extrinsic properties such as surface chemistry and aggregation that depend on the environment and determine the fate and reactivity of the material (“where they go” and “what they do”). Methods are typically optimized for pristine ENM and may not be applicable to the required environmental or use conditions. In other cases validated protocols are missing or rarely used by the community. Second, many ENM properties are method-defined. For example, particle size can be assessed by more than ten different methods, each with its own range of applicability, theoretical basis and sensitivity. Particle size distributions may be based on intensity, number or mass and can be measured by either ensemble methods that interrogate the entire sample or by particle counting methods such as microscopy techniques. Selection of a fit-for-purpose method must consider the theoretical basis of the technique, method sensitivity and calibration, analyte concentration, sample matrix, potential ENM transformations, and the acceptable uncertainty for a specific application.

Despite this complexity, there is an emerging consensus on the key properties that are necessary for risk assessment and how they should be measured. There is also a growing appreciation that the dynamic nature of nanomaterials and the high potential for batch-to-batch variability necessitate careful data reporting. However, significant bottlenecks remain in several areas. Although it is currently feasible to measure most properties of the pristine material, it is much more challenging to detect and characterize ENM in complex environmental and toxicological media where the concentration is low and there is interference from components or contaminants. Agglomeration and/ or aggregation of the ENM varies with time, medium and particle concentration and is considerably more problematic for “real-world” ENM than for the well-behaved, spherical, and monodisperse materials typically used for method development. Methods to determine the aggregation state of the material in the medium of interest are infrequently applied and are time- and resource-intensive. A final consideration is the difficulty with reproducible functionalization of materials and quantification of surface groups and coatings. Surface chemistry controls the fate and behaviour of a nanomaterial when incorporated into products, released to the environment or taken up by cells or organisms.

Figure. 1. Illustration of some of the ENM properties that must be characterized, reproduced with permission [1]

Transformations of ENM in the environment and consumer products

The distribution and transformations of ENM in environmental matrices are determined by properties such as surface charge and hydrophobicity, solubility, aggregation state and chemical reactivity. Possible transformations in matrices such as soil or water include dissolution (which means that effects of both the ENM and dissolved ions must be considered) and redox processes which can alter the chemical reactivity of the material (Figure 2). Aggregation, sedimentation, and adsorption on matrix components such as natural organic matter in soil are also commonly observed. ENM transformations are modulated by the properties of the matrix (e.g., pH, ionic strength, other components or contaminants) and will alter the transport and bioavailability of the ENM. The low ENM concentration and the requirement to distinguish it from other naturally occurring nanomaterials make the detection and characterization of the ENM in environmental matrices a difficult problem. Transformations of ENM in complex environmental media hinder the determination of appropriate exposure levels for assessing toxicity. Nevertheless, the interaction of ENM with environmental matrices under realistic exposure conditions does not necessarily increase reactivity, bioavailability or toxicity, suggesting that the impact of ENM on ecosystems may frequently be lower than anticipated.

Figure. 2. Illustration of possible transformations of metal oxide nanoparticles in an environmental sample, reproduced with permission [1].

Assessing the consequences of ENM release from consumer products requires a careful life-cycle analysis that accounts for the method of ENM incorporation, the possible uses of the product and the disposal scenarios at end-of-use. For example, if an ENM is used to improve the mechanical properties of a polymer composite, one must consider the release of the unmodified or matrix-modified ENM from the composite and the potential toxicity of both forms, as well as any changes in the toxicity or degradability of the matrix caused by the ENM. ENM release tests often use a tiered approach that begins with the pristine material in a controlled environment where the effects of specific variables can be readily addressed and then progresses to studies of the product under normal use conditions. The difficulty in obtaining quantitative analytical data on ENM release from consumer products has motivated the development of exposure modeling to predict environmental concentration. Models focus on either tracking the flow of the ENM from production to end-of-life or assessing the distribution of ENMs between various environmental compartments. Integration of the two approaches has significant potential, but is still limited by the lack of life-cycle analysis data to validate the models.

Nanotoxicology methods and gaps for environmental health and safety

In vitro assays are widely used in nanotoxicology but comparisons between studies are hampered by inadequate characterization of the ENM, insufficient consideration of the biologically relevant dose, and failure to use standardized methods and controls. As noted above the characterization issue is easy to solve, although potentially resource intensive. The question of dose is more problematic; many studies use ENM doses that far exceed the anticipated exposure level in order to see a biological effect. More sensitive techniques for determining biological endpoints are needed to facilitate use of more relevant doses. There is a lack of consensus on whether mass, surface area or particle number is the most appropriate dose metric and all three metrics are complicated by the propensity of ENM to aggregate, sediment or dissolve on the exposure time scale. Approaches to disperse ENM reproducibly, control or monitor their agglomeration state and measure cellular uptake are necessary to ensure that nanotoxicology experiments assess realistic exposure conditions.

The rapidly increasing number of new ENM and the desire to minimize animal studies provide a strong impetus for extensive use of in vitro tests. Important considerations include the use of multiple assays for a given endpoint, inclusion of controls, consideration of possible ENM interference and validation of the assay. Validated protocols for some cytotoxicity assays have been developed. Application of more realistic models such as primary cell lines, 3D cellular models and co-cultures will improve the utility of in vitro tests for predicting in vivo behaviour. The need to minimize testing has also led to the development of grouping and read across methods for nanomaterial assessment. The goal is to determine if data for one nanoform can be applied to related materials and the level of data needed to support such a decision. Successful development of grouping/read across methods will require data from validated characterization and toxicology assay protocols and will be facilitated by standard reporting practices, accessible data bases and computational and machine learning tools.

Challenges for standardization and risk assessment

Continued development of standardized, internationally validated methods is required to address many of the above issues with ENM characterization and toxicology assays. A number of large multi-laboratory projects are addressing this gap by developing and testing tools to assess nanomaterial safety. Integration of such activities with ongoing work at standardization organizations such as ISO will lead to improved harmonization and efficiency. Future standardization efforts must provide sample preparation protocols and analytical methods that are relevant to the complex matrices in which ENM fate and behaviour must be assessed. Reference materials with complex sizes and shapes and high polydispersity are essential to develop methods and protocols that must then be validated in interlaboratory comparisons. The development of biologically relevant exposure limits reinforces the need for improved detection of ENM in complex environments, including workplace scenarios. Progress in these area will allow the development of occupational exposure limits and the design of regulatory frameworks for use of nanomaterials.

Summary and future perspectives

The main gaps and challenges highlighted above are summarized in Table 1 (see full text PDF), along with research priorities that offer potential solutions. Advances in these areas will enhance our ability to assess and ultimately predict nanomaterial properties and behaviour in different environments. This will facilitate more efficient and cost-effective methods for evaluating the safety of ENM and developing regulatory guidelines for their use. Several current IUPAC projects are providing guidance that is necessary for the safe and sustainable application of nanotechnology. These include projects on Human Health Risk Considerations of Nano-enabled Pesticides for Industry and Regulators (2017-035-2-600), Analytical Chemistry of Nanomaterials—Critical Evaluation (2017-005-3-500), and Nomenclature and Associated Terminology for Inorganic Nanoscale Particles (2019-016-3-800).

Reference 1. L. J. Johnston, N. Gonzalez-Rojano, K. J. Wilkinson, B. Xing, Key challenges for evaluation of the safety of engineered nanomaterials, NanoImpact, 18, 100219, 2020.

Acknowledgements

This work was supported by IUPAC project 2016- 045-2-700. We thank the workshop invited speakers, participants and industrial exhibitors whose contributions formed the initial motivation for the NanoImpact article [1] and this summary.

Linda J. Johnston, National Research Council Canada, Ottawa, Canada, Norma Gonzalez-Rojano, Centro Nacional de Metrologia, Queretaro, Mexico, Kevin J. Wilkinson, Université de Montréal, Montréal, Canada, Baoshan Xing, Stockbridge School of Agriculture, University of Massachusetts, Amherst, USA

Cite: Johnston, L. J., Gonzalez-Rojano, N., Wilkinson, K. J., & Xing, B. (2021). Challenges for Evaluation of the Safety of Engineered Nanomaterials, Chemistry International, 43(1), 4-7; https://doi.org/10.1515/ci-2021-0102