Jutta Tentschert

Nanomaterial categorization for assessing risk potential to facilitate regulatory decision-making

For nanotechnology to meet its potential as a game-changing and sustainable technology, it is important to ensure that the engineered nanomaterials and nanoenabled products that gain entry to the marketplace are safe and effective. Tools and methods are needed for regulatory purposes to allow rapid material categorization according to human health and environmental risk potential, so that materials of high concern can be targeted for additional scrutiny, while material categories that pose the least risk can receive expedited review. Using carbon nanotubes as an example, we discuss how data from alternative testing strategies can be used to facilitate engineered nanomaterial categorization according to risk potential and how such an approach could facilitate regulatory decision-making in the future.

Application of Laser Postionization Secondary Neutral Mass Spectrometry/Time-of-Flight Secondary Ion Mass Spectrometry in Nanotoxicology: Visualization of Nanosilver in Human Macrophages and Cellular Responses

Silver nanoparticles (SNP) are the subject of worldwide commercialization because of their antimicrobial effects. Yet only little data on their mode of action exist. Further, only few techniques allow for visualization and quantification of unlabeled nanoparticles inside cells. To study SNP of different sizes and coatings within human macrophages, we introduce a novel laser postionization secondary neutral mass spectrometry (Laser-SNMS) approach and prove this method superior to the widely applied confocal Raman and transmission electron microscopy. With time-of-flight secondary ion mass spectrometry (TOF-SIMS) we further demonstrate characteristic fingerprints in the lipid pattern of the cellular membrane indicative of oxidative stress and membrane fluidity changes. Increases of protein carbonyl and heme oxygenase-1 levels in treated cells confirm the presence of oxidative stress biochemically. Intriguingly, affected phagocytosis reveals as highly sensitive end point of SNP-mediated adversity in macrophages. The cellular responses monitored are hierarchically linked, but follow individual kinetics and are partially reversible.

A medical-toxicological view of tattooing

Long perceived as a form of exotic self-expression in some social fringe groups, tattoos have left their maverick image behind and become mainstream, particularly for young people. Historically, tattoo-related health and safety regulations have focused on rules of hygiene and prevention of infections. Meanwhile, the increasing popularity of tattooing has led to the development of many new colours, allowing tattoos to be more spectacular than ever before. However, little is known about the toxicological risks of the ingredients used. For risk assessment, safe intradermal application of these pigments needs data for toxicity and biokinetics and increased knowledge about the removal of tattoos. Other concerns are the potential for phototoxicity, substance migration, and the possible metabolic conversion of tattoo ink ingredients into toxic substances. Similar considerations apply to cleavage products that are formed during laser-assisted tattoo removal. In this Review, we summarise the issues of concern, putting them into context, and provide perspectives for the assessment of the acute and chronic health effects associated with tattooing.

Toxicity of silver nanoparticles in human macrophages: uptake, intracellular distribution and cellular responses

Silver nanoparticles (SNP) are among the most commercialized nanoparticles worldwide. They can be found in many diverse products, mostly because of their antibacterial properties. Despite its widespread use only little data on possible adverse health effects exist. It is difficult to compare biological data from different studies due to the great variety in sizes, coatings or shapes of the particles. Here, we applied a novel synthesis approach to obtain SNP, which are covalently stabilized by a small peptide. This enables a tight control of both size and shape. We applied these SNP in two different sizes of 20 or 40 nm (Ag20Pep and Ag40Pep) and analyzed responses of THP-1-derived human macrophages. Similar gold nanoparticles with the same coating (Au20Pep) were used for comparison and found to be non-toxic. We assessed the cytotoxicity of particles and confirmed their cellular uptake via transmission electron microscopy and confocal Raman microscopy. Importantly a majority of the SNP could be detected as individual particles spread throughout the cells. Furthermore we studied several types of oxidative stress related responses such as induction of heme oxygenase I or formation of protein carbonyls. In summary, our data demonstrate that even low doses of SNP exerted adverse effects in human macrophages.

State of the art in human risk assessment of silver compounds in consumer products: a conference report on silver and nanosilver held at the BfR in 2012

In light of the broad spectrum of products containing nanosilver, the harmfulness of nanosilver to human health and the environment was intensively discussed at a conference held in February 2012 at the BfR. The conference agenda covered the aspects of analytics of nanosilver materials, human exposure and toxicology as well as effects on microorganisms and the environment. The discussion recovered major gaps related to commonly agreed guidelines for sample preparation and central analytical techniques. In particular, the characterization of the nanoparticles in complex matrices was regarded as a challenge which might become a pitfall for further innovation and application. Historical and anecdotal records of colloidal silver have been sometimes taken as empirical proof for the general low toxicity of nanosilver. Yet as reported herein, a growing number of animal studies following modern performance standards of toxicity testing have been carried out recently revealing well-characterized adverse effects on different routes of exposure in addition to argyria. Furthermore, recent approaches in exposure assessment were reported. However, consumer exposure scenarios are only starting to be developed and reliable exposure data are still rare. It was further widely agreed on the workshop that the use of silver may lead to the selection of silver resistant bacteria. With respect to its environmental behavior, it was suggested that nanosilver released to wastewater may have negligible ecotoxicological effects. Finally, the presentations and discussion on risk assessment and regulation of nanosilver applications gave insights into different approaches of risk assessment of nanomaterials to be performed under the various regulatory frameworks.

Risk assessment of nanomaterials in cosmetics: a European union perspective

In Europe, the data requirements for the hazard and exposure characterisation of chemicals are defined according to the REACH regulation and its guidance on information requirements and chemical safety assessment (Regulation (EC) No 1907/2006 of the European Parliament and of the Council of 18 December 2006 concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), and its guidance documents; available at: http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2006:396:0001:0849:EN:PDF; and at: http://guidance.echa.europa.eu/docs/guidance_document/information_requirements_en.htm). This is the basis for any related risk assessment. The standard reference for the testing of cosmetic ingredients is the SCCP’s ‘Notes of Guidance for the Testing of Cosmetic Ingredients and their Safety Evaluation’ (The SCCP’s Notes of Guidance for the testing of cosmetic ingredients and their safety evaluation (2006); available at: http://ec.europa.eu/health/ph_risk/committees/04_sccp/docs/sccp_o_03j.pdf), which refers to the OECD guidelines for the testing of chemicals (The OECD Guidelines for the Testing of Chemicals as a collection of the most relevant internationally agreed testing methods used by government, industry and independent laboratories to assess the safety of chemical products; available at: http://www.oecd.org/topic/0,2686,en_2649_34377_1_1_1_1_37407,00.html). According to the cosmetics directive [76/768/EEC], compounds that are classified as mutagenic, carcinogenic or toxic to reproduction are banned for the use in cosmetic products. Since December 2010, the respective labelling is based on the rules of regulation (EC) No. 1272/2008 (Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006, Official Journal L 353, 31/12/2008, pages 1–1355; available at: http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2008:353:0001:1355:en:PDF) on classification, labelling and packaging of substances and mixtures (CLP). There is no further impact from the CLP regulation on cosmetic products, because regulation (EC) No. 1223/2009 on cosmetic products defines its own labelling rules (Regulation (EC) No 1223/2009 of the European Parliament and of the Council of 30 November 2009 on cosmetic products; available at: http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2009:342:0059:0209:en:PDF). Special notification procedures are mandatory for preservatives, colourants and UV-filters where a safety approval from the European ‘Scientific Committee on Consumer Safety’ (SCCS) is needed prior to marketing. The risk assessment of nanomaterials in consumer products still poses a significant challenge as highlighted by the example of UV-filters in sunscreens since nanomaterials cannot be classified as a homogenous group of chemicals but still need to be addressed in risk characterisation on a case by case basis.

Biokinetics of nanomaterials: The role of biopersistence

Nanotechnology risk management strategies and environmental regulations continue to rely on hazard and exposure assessment protocols developed for bulk materials, including larger size particles, while commercial application of nanomaterials (NMs) increases. In order to support and corroborate risk assessment of NMs for workers, consumers, and the environment it is crucial to establish the impact of biopersistence of NMs at realistic doses. In the future, such data will allow a more refined future categorization of NMs. Despite many experiments on NM characterization and numerous in vitro and in vivo studies, several questions remain unanswered including the influence of biopersistence on the toxicity of NMs. It is unclear which criteria to apply to characterize a NM as biopersistent. Detection and quantification of NMs, especially determination of their state, i.e., dissolution, aggregation, and agglomeration within biological matrices and other environments are still challenging tasks; moreover mechanisms of nanoparticle (NP) translocation and persistence remain critical gaps. This review summarizes the current understanding of NM biokinetics focusing on determinants of biopersistence. Thorough particle characterization in different exposure scenarios and biological matrices requires use of suitable analytical methods and is a prerequisite to understand biopersistence and for the development of appropriate dosimetry. Analytical tools that potentially can facilitate elucidation of key NM characteristics, such as ion beam microscopy (IBM) and time-of-flight secondary ion mass spectrometry (ToF-SIMS), are discussed in relation to their potential to advance the understanding of biopersistent NM kinetics. We conclude that a major requirement for future nanosafety research is the development and application of analytical tools to characterize NPs in different exposure scenarios and biological matrices.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS)-based analysis and imaging of polyethylene microplastics formation during sea surf simulation.

Plastic particles smaller than 5mm, so called microplastics have the capability to accumulate in rivers, lakes and the marine environment and therefore have begun to be considered in eco-toxicology and human health risk assessment. Environmental microplastic contaminants may originate from consumer products like body wash, tooth pastes and cosmetic products, but also from degradation of plastic waste; they represent a potential but unpredictable threat to aquatic organisms and possibly also to humans. We investigated exemplarily for polyethylene (PE), the most abundant constituent of microplastic particles in the environment, whether such fragments could be produced from larger pellets (2mm×6mm). So far only few analytical methods exist to identify microplastic particles smaller than 10μm, especially no imaging mass spectrometry technique. We used at first time-of-flight secondary ion mass spectrometry (ToF-SIMS) for analysis and imaging of small PE-microplastic particles directly in the model system Ottawa sand during exposure to sea surf simulation. As a prerequisite, a method for identification of PE was established by identification of characteristic ions for PE out of an analysis of grinded polymer samples. The method was applied onto Ottawa sand in order to investigate the influence of simulated environmental conditions on particle transformation. A severe degradation of the primary PE pellet surface, associated with the transformation of larger particles into smaller ones already after 14days of sea surf simulation, was observed. Within the subsequent period of 14days to 1month of exposure the number of detected smallest-sized particles increased significantly (50%) while the second smallest fraction increased even further to 350%. Results were verified using artificially degraded PE pellets and Ottawa sand.

Impact of an Artificial Digestion Procedure on Aluminum-Containing Nanomaterials

Aluminum has gathered toxicological attention based on relevant human exposure and its suspected hazardous potential. Nanoparticles from food supplements or food contact materials may reach the human gastrointestinal tract. Here, we monitored the physicochemical fate of aluminum-containing nanoparticles and aluminum ions when passaging an in vitro model of the human gastrointestinal tract. Small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), ion beam microscopy (IBM), secondary ion beam mass spectrometry (TOF-SIMS), and inductively coupled plasma mass spectrometry (ICP-MS) in the single-particle mode were employed to characterize two aluminum-containing nanomaterials with different particle core materials (Al0, γAl2O3) and soluble AlCl3. Particle size and shape remained unchanged in saliva, whereas strong agglomeration of both aluminum nanoparticle species was observed at low pH in gastric fluid together with an increased ion release. The levels of free aluminum ions decreased in intestinal fluid and the particles deagglomerated, thus liberating primary particles again. Dissolution of nanoparticles was limited and substantial changes of their shape and size were not detected. The amounts of particle-associated phosphorus, chlorine, potassium, and calcium increased in intestinal fluid, as compared to nanoparticles in standard dispersion. Interestingly, nanoparticles were found in the intestinal fluid after addition of ionic aluminum. We provide a comprehensive characterization of the fate of aluminum nanoparticles in simulated gastrointestinal fluids, demonstrating that orally ingested nanoparticles probably reach the intestinal epithelium. The balance between dissolution and de novo complex formation should be considered when evaluating nanotoxicological experiments.

A knowledge-based search engine to navigate the information thicket of nanotoxicology

The risk assessment of nano-sized materials (NM) currently suffers from great uncertainties regarding their putative toxicity for humans and the environment. An extensive amount of the respective original research literature has to be evaluated before a targeted and hypothesis-driven Environmental and Health Safety research can be stipulated. Furthermore, to comply with the European animal protection legislation in vitro testing has to be preferred whenever possible. Against this background, there is the need for tools that enable producers of NM and risk assessors for a fast and comprehensive data retrieval, thereby linking the 3Rs principle to the hazard identification of NM. Here we report on the development of a knowledge-based search engine that is tailored to the particular needs of risk assessors in the area of NM. Comprehensive retrieval of data from studies utilising in vitro as well as in vivo methods relying on the PubMed database is presented exemplarily with a titanium dioxide case study. A fast, relevant and reliable information retrieval is of paramount importance for the scientific community dedicated to develop safe NM in various product areas, and for risk assessors obliged to identify data gaps, to define additional data requirements for approval of NM and to create strategies for integrated testing using alternative methods.