Grouping, Read-Across, CharacterIsation and classificatiOn framework and strategies for regUlatory risk assessment of manufactured nanomaterials and Safer design of nano-enabled products (GRACIOUS)

Manufacturing and functionalising materials at the nanoscale leads to a whole array of nanomaterials (NMs) or nanoforms (NFs) varying in e.g. size, morphology and surface characteristics. Safety assessment requires sufficient information for each NF, but testing every unique material variant for all potential adverse effects is virtually impossible. More efficient ways for safety assessment are therefore needed, e.g. by applying grouping and read-across approaches. On a conceptual level, regulatory accepted approaches for grouping and read-across for NMs already existed at the start of the project, but their actual application remained difficult for various reasons. Therefore, the overarching goal of GRACIOUS was to generate an innovative, science-based Framework to enable practical application of grouping and read-across of NMs/NFs. To achieve this, GRACIOUS integrated key stakeholder needs with state-of-the-art scientific knowledge. GRACIOUS developed grouping hypotheses, criteria, guiding principles and refined/ integrated tools. The GRACIOUS Grouping Framework was practically applied and tested in a series of case studies.

GRACIOUS developed a comprehensive, innovative, science-based Framework to enable practical application of grouping and read-across of nanomaterials (NMs)/nanoforms (NFs). The framework was initially released in a draft version, discussed with stakeholders and edited based upon their feedback and published as an improved version (Stone et al.short foret alii (lat. "and others") 2020). In addition, grouping hypotheses were developed and refined during the course of the project. Each of the grouping hypothesis was supported by an integrated approach to testing and assessment (IATA), which guides the user to compile the relevant information in a decision-tree manner. Where necessary sub-hypotheses were formulated to ensure clear IATAs. In total about 19 (sub‑)hypotheses were finalised for human health and about 22 (sub‑)hypotheses for the environment. Importantly, the GRACIOUS IATAs are modular. Several generic decision nodes such as dissolution or reactivity are included in several IATAs and can also be used to generate additional user-specific IATAs.The BfRshort forGerman Federal Institute for Risk Assessment led the testing of the GRACIOUS grouping framework in case studies within Task 1.6 including several project-internal case studies conducted in close collaboration between WP4 and WP5. Those included different grouping purposes (i.e. regulatory and Safe-by-Design), exposureClose dialogexposureExposureTo glossary routes, environmental compartments and types of NF. The case studies were prioritized for NFs with plenty of data available to reduce the need to generate new data. Four inhalation case studies addressed grouping of MWCNT, carbon black, SiO2 or pigments. One oral case study focused on different SiO2 NF. Environmental case studies investigated coated nanoforms, looking at environmental degradation of surface coatings, as well as toxicity (related to the core chemistry and size) to soil bacteria and phytotoxicity. Some case studies addressed individual decision nodes only such as dissolution or reactivity. Based on stakeholder feedback, another case study was included to develop a IATA for genotoxicity of NFs. External stakeholders also contributed to testing the framework and conducted six additional case studies.GRACIOUS published several manuscripts on the IATAs and the case studies. The BfRshort forGerman Federal Institute for Risk Assessment contributed to several of them on a conceptual level. The experimental work at the BfRshort forGerman Federal Institute for Risk Assessment primarily focused on NF reactivity and cellular oxidative stress. Reactivity and oxidative stress were identified as important grouping parameters, often contributing to the toxicity of NFs. Hence they were considered in several GRACIOUS IATAs. NF reactivity and oxidative stress can be assessed using a number of approaches but before GRACIOUS there was little systematic work. GRACIOUS designed a tiered testing strategy including the assessment of NF reactivity (i.e. ROS generation) in acellular conditions in a first tier and suggested a combination of different cell-based assays to detect whether NMs cause oxidative stress in different cell models in a second tier. GRACIOUS reached all its objectives and developedn a practically applicable Framework for the grouping of NMs/NFs. Selected publications with BfRshort forGerman Federal Institute for Risk Assessment involvement:1) Stone V., Gottardo S., Bleeker E.A.J., Braakhuis H., Dekkers S., Fernandes T., Haase A., Hunt N., Hristozov D., Jantunen P., Jeliazkova N., Johnston H., Lamon L., Murphy F., Rasmussen K., Rauscher H., Jiménez A.S., Svendsen C., Spurgeon D., Vázquez-Campos S., Wohlleben W., and Oomen A.G. (2020): A framework for grouping and read-across of nanomaterials- supporting innovation and risk assessment. Nano Today 35, 100941. DOI: https://doi.org/10.1016/j.nantod.2020.100941 
2) Gimeno-Benito I., Giusti A., Dekkers S., Haase A., and Janer G. (2021): A review to support the derivation of a worst-case dermal penetration value for nanoparticles. Regulatory Toxicology and Pharmacology 119, 104836. DOI: https://doi.org/10.1016/j.yrtph.2020.104836   3) Murphy F., Dekkers S., Braakhuis H., Ma-Hock L., Johnston H., Janer G., di Cristo L., Sabella S., Jacobsen N.R., Oomen A.G., Haase A., Fernandes T., and Stone V. (2021): An integrated approach to testing and assessment of high aspect ratio nanomaterials and its application for grouping based on a common mesothelioma hazard. NanoImpact 22, 100314. DOI: https://doi.org/10.1016/j.impact.2021.100314 
4)  Di Cristo L., Oomen A.G., Dekkers S., Moore C., Rocchia W., Murphy F., Johnston H.J., Janer G., Haase A., Stone V., Sabella S. (2021): Grouping hypotheses and an Integrated Approach to Testing and Assessment of Nanomaterials Following Oral Ingestion. Nanomaterials 11 (10), 2623. DOI: https://doi.org/10.3390/nano11102623  
5) Jeliazkova N., Bleeker E., Cross R., Haase A., Janer G., Peijnenburg W., Pink M., Rauscher H., Svendsen C., Tsiliki G., Zabeo A., Hristozov D., Stone V., and Wohlleben W. (2022): How can we justify grouping of nanoforms for hazard assessment? Concepts and tools to quantify similarity. NanoImpact 25, 100366. DOI: https://doi.org/10.1016/j.impact.2021.100366 
6) Ag Seleci D., Tsiliki G., Werle K., Elam D.A., Okpowe O., Seidel K., Bi X., Westerhoff P., Innes E., Boyles M., Miller M., Giusti A., Murphy F., Haase A., Stone V., and Wohlleben W. (2022): Determining nanoform similarity via assessment of surface reactivity by abiotic and in vitro assays. NanoImpact 26, 100390. DOI: https://doi.org/10.1016/j.impact.2022.100390 
7) Verdon R., Stone V., Murphy F., Christopher E., Johnston H. J., Doak S. H., Vogel U., Haase A.,  and Kermanizadeh A. (2022): The application of existing genotoxicity methodologies for grouping of nanomaterials: towards an integrated approach to testing and assessment. Particle and Fibre Toxicology 19 (1), 32. DOI: https://doi.org/10.1186/s12989-022-00476-9 
8) Di Cristo L., Janer G., Dekkers S., Boyles M., Giusti A., Keller J.G., Wohlleben W., Braakhuis H., Ma-Hock L., Oomen A.G., Haase A., Stone V., Murphy F., Johnston H.J., and Sabella S. (2022): Integrated approaches to testing and assessment for grouping nanomaterials following dermal exposure. Nanotoxicology 16 (3), 310-332. DOI: https://doi.org/10.1080/17435390.2022.208520 9) Boyles M., Murphy F., Mueller W., Wohlleben W., Jacobsen N.R., Braakhuis H., Giusti A., and Stone V. (2022): Development of a standard operating procedure for the DCFH2-DA acellular assessment of reactive oxygen species produced by nanomaterials. Toxicology Mechanisms and Methods 32 (6), 439-452. DOI: https://doi.org/10.1080/15376516.2022.2029656