As the NanoFASE project draws to a close (Sept 2019) we share information on how our work and several of our lead investigators have contributed to standardization efforts at international and national levels. Of particular note: the contributions to procedures and guidance on assessing environmental transformations of nanomaterials, heteroaggregation and dissolution kinetics, leaching in soil column, and a tiered approach to bioaccumulation.

OECD TG/GD

NanoFASE’s contributions to OECD Technical Guidelines and Guidance Documents are captured in the OECD reports on the Developments in Delegations on the Safety of Manufactured Nanomaterials - Tour de Table from 2017-2019.  The latest report published in May 2019 can be accessed here. Highlighted are:

• NanoFASE activities towards OECD TG/GD development and the remarkable engagement by UNIVIE under the leadership of Frank von der Kammer.
• Work lead by Wageningen University and Research (WUR) on biota uptake of nanomaterials where exposure and uptake studies have been initiated in earthworms and in in vitro models for the human gastrointestinal tract (RIKILT). 
• RIVM’s contribution to the development of algorithms for quantifying the fate of nanomaterials in the aquatic environment, and the update of the SimpleBox4nano model using NanoFASE output. This update is aimed at direct application in regulatory frameworks. 
• Environmental exposure tools from NanoFASE and their regulatory relevance as discussed at ECHA’s Nanomaterial Expert Group.
• The project results to be input to the current efforts in nanospecific modification of the OECD guidance documents on testing of chemicals.

In February 2018, NanoFASE put forward New Project Proposals for TG/GD related work under the OECD WPMN guidelines revision projects. Several activities were initiated and on-going activities were fastened to reach as far as possible within the project lifetime.

UNIVIE, Vienna under the leadership of Frank van der Kammer, WP8 NanoFASE, led the following activities:

• New GD on Environmental Transformation: UNIVIE led the NanoFASE work on Transformation/Dissolution in environmental media. The procedures developed in NanoFASE for solubility/dissolution and the media compositions used for heteroagglomeration inform the development of the new OECD GD on Environmental Transformation of Nanomaterials and support harmonization between the transformation tests (developed by UNIVIE), the dissolution in water and biological media tests (DK2)(NRCWE/BfR) and the new activity for dissolution in environmental media (UBA), which will lead to nanospecific guidance documents or TGs.
• NanoFASE work on heteroagglomeration supports the introduction of heteroagglomeration into TG 318 Dispersion Stability of Nanomaterials in Simulated Environmental Media via additions to the Guidance Document (OECD project 3.10), removing the need to develop a new TG. Here too UNIVIE supported OECD ensuring NanoFASE knowledge is directly incorporated to the OECD process. In February 2019, UNIVIE together with the Environmental Protection Agency of Germany organized a writing workshop to move the GD forward with regard to dissolution and heteroagglomeration. Members of NanoFASE contributed to the writing of the GD during this workshop.
  - Hetero aggregation Tier 1- screening for a wider particle number with an output as high, medium, low
  - Hetero aggregation Tier 2 - detailed, output in the form of alpha-values
• Dissolution kinetics Tier 1- screening - Would address Denmark I and GD on dissolution in water and biological media (DK 2) and future TG on dissolution in environmental media (dynamic method) (GER)
• Dissolution kinetics Tier 2- dissolution rate - Would address Denmark I and GD on dissolution in water and biological media (DK 2) and future TG on dissolution in environmental media (dynamic method) (GER)

SLU, Sweden under the leadership of Geert Cornelis, WP7 NanoFASE, led the following activities:

• Test No. 312: Leaching in Soil Column: SLU updated the guidance document and obtained further review from NanoFASE partners from both US and Europe. Contributions include the testing of two nanomaterials (Ag and CeO2) in different soils, which lends insight on the robustness of the protocol and determines if any further changes are needed. Duke University and the NanoCommons project were contacted for the storage of data originating from this protocol. Geert Cornelis attended the December 2018 expert meeting to discuss all the projects related to Ecotoxicity and Fate of Nanomaterials to foresee the development of this guidance document.
• Test No. 106: Adsorption -- Desorption Using a Batch Equilibrium Method: SLU is also contributing towards this in collaboration with Duke University. Discussions are on-going.

UoP, UK under the leadership of Richard Handy, WP9 NanoFASE, led the following activities:

• TG305 and establishing a tiered approach to bioaccumulation testing. Handy et. al were especially engaged with the WPMN at the OECD on the bioaccumulation testing of nanomaterials. Currently, the in vivo fish test TG305 is mandatory and there are no in vitro steps before that. Our strategy for the OECD is described in detail in a scientific paper, as well as in draft OECD documents. The strategy involves a first tier on ‘chemical triggers of concern’ that are relevant to nanomaterials to start the process, such as dissolution kinetics and settling rates. However, our focus has been on establishing a ‘new’ ex-vivo protocol involving fish gut sacs that enables the bioaccumulation potential to be predicted (or excluded), such that only materials that show the bioaccumulation potential go forward to the lengthy and expensive in vivo testing with fish. The gut sac studies will be tier 3 of the strategy, and the final in vivo testing (TG305) as tier 4. NanoFASE data has contributed data in three ways: (i) demonstration/validation of the gut sac method (Clark et al., 2019a) as an early tier that could help screen out materials of low concern, and also to reduce the need for using the in vivo tests (3Rs, reducing and replacing animal testing). (ii) A further demonstration that the dietary exposure method in TG305 also works for nanomaterials (Clark et al. 2019b). This latter paper is an in depth in vivo study of silver materials, and complements the previously done work on other EU projects at Plymouth on dietary exposure to nanomaterials. Finally, (iii) to broaden the data sets, more gut sac studies were performed  in the last few months of NanoFASE with materials that represent a range of chemistries (metal/metal oxides, carbon-based, etc). This data set can be used to demonstrate the tiered approach to the OECD in technical workshops during 2019-2020. This OECD work is ongoing, and hopefully will be drawn into the second stage of the Malta Initiative.
• Bioaccumulation studies are sometimes required for biomedical applications. There are a number of repeated-dose oral toxicity tests with rodents such as OECD TG407. There are also OECD studies intended to inform on uptake in mammals, usually with rodents such as TG 417: Toxicokinetics. The concept of replacing these in vivo rodent tests by an ex-vivo tier also applies, and in NanoFASE the rodent gut sac was also used. There is potential to reduce both rodent and fish testing with the tiered approach and methods therein. A publication on the rodent work is in progress, as well as papers demonstrating particles inside the tissues by single particle ICP-MS. This latter strand of work is also helping colleagues at Public Health England in the UK establish methods for nanomaterials in food, as well as Defra in the UK on the environmental aspects. Work with both these UK agencies is ongoing.

NanoFASE Involvement in European and National Committees

Christof Asbach (IUTA), NanoFASE WP3 leader and European co-chair of the US-EU community of research (CoR) for "Exposure along the product life") is an active participant in several standardisation groups:

• CEN TC137/WG3 "Workplace Exposure - Particulate Matter" and its mirror group ‘The German Institute for Standardization’ (DIN AK Staub): Lessons learned from NanoFASE fed into different standards here, including a standard on metrics to be used for assessing nanoparticle exposure in workplaces, on dustiness and on determining the size weighted fine fraction (SWFF) of respirable silica. The contribution is of general nature, because the experiments conducted in NanoFASE are too specific to be used in the standards. Christof Asbach also contributed to the meeting held in Warsaw in June 2019 for the further development of CEN TC137/WG3.
• The Association of German Engineers (VDI Group) "Measurement of the particulate number concentration in the environment": The group has developed several VDI guidelines in the last few years, where IUTA contributed knowledge gained from the NanoFASE experiments. As above, these are rather general in nature, e.g. on the generation of nanoparticle aerosols with different methods and their measurement by e.g. condensation particle counters, electrical mobility analysers as well as particle sampling. Currently, a guideline on particle sampling and transport is under development, where information gained from the construction of our test rig feeds in regarding the effect of TiO2 nanoparticles on atmospheric NO2.

 

NanoFASE WP9 deputy leader Susana Loureiro (UVAR – PT) participates in the Portuguese national technical commission for Nanotechnologies, related to the ISO guidelines.

Within the proposals for new working documents from Slovenia, NanoFASE partners from University of Ljubljana are members of CEN/TC 352 - Nanotechnologies and submitted a proposal (NanoFASE WP9) on: Feeding exposure of terrestrial crustaceans (Porcellio scaber, Isopoda) to study the bioaccumulation of metals dissolved from nanomaterials. Such work for environmental species is important for key PBT (Persistent, Bioaccumulative and Toxic) and vPvB (very Persistent and very Bioaccumulative) substance properties under REACH.

Malta Initiative

The Malta Initiative is a joint RTD – Member States initiative to increase EU presence and contribution on adapting and developing new nano-specific OECD testing guidelines and guidance documents for nanomaterials. Revision of REACH Annexes for nanomaterials will be in alignment with the update/development in OECD. The NanoFASE standardisation activities are contributing to the Malta Initiative. A Malta Initiative workshop was organised in December 2018 to bring together relevant knowledge, testing methods and harmonised standards gathered and developed in several EU-funded nanosafety research projects. Iseult Lynch, Nanosafety Cluster, UK and NanoFASE WP3 leader presented ‘Enabling FAIRness and Openness of EU Nanosafety cluster data – the NanoCommons approach’. Claus Svendsen (NERC-CEH), Richard Handy (UoP) also attended and participated in discussions around tiered approaches for bioaccumulation, and broader aspects relating to the NanoFASE project.