A recent review discusses the suitability of in vitro models for studying the intestinal uptake of nanomaterials. While Caco-2 cell models are widely recognised for studying chemical uptake, their suitability for nanomaterials is limited due to the complex physiological processes involved, prompting the need for more advanced co-culture models. Significant knowledge gaps remain, especially in standardising and assessing how well these models mimic human biology and relevant exposure scenarios. Currently, the first steps towards harmonization of new approach methodologies as a tool to predict intestinal uptake of nanomaterials are being taken.

The importance of understanding the intestinal fate of nanomaterials

A recent review discusses the evolving landscape of New Approach Methodologies (NAMs) for studying the intestinal fate of nanomaterials (NMs) following oral ingestion. As NMs are incorporated in food additives and packaging to enhance product quality, safety and stability, it is important to understand their impact on human health. The intestinal uptake of NMs is critical for assessing their systemic effects. The intestine serves as a biological barrier, protecting the human body by preventing toxic substances and pathogens from entering the bloodstream.

Differences in intestinal uptake between nanomaterials and chemicals

The human intestinal cell line Caco-2, grown on a permeable insert, is the standard model used to study the uptake of chemicals, as recognised by organisations such as the OECD, the European Medicines Agency (EMA) and the Food and Drug Administration (FDA). However, while these models have proven valuable for studying chemicals, their suitability for NMs is less clear, given the complexities of NM uptake, which involves multiple cell types and physiological processes.

Evaluation of NMs after ingestion: challenges and insights

The review examines various categories of NMs relevant for oral exposure. It provides a comprehensive overview of existing literature regarding their uptake, ability to cross the intestinal barrier, and potential to impair intestinal barrier functionality. The review also included in vitro digestion experiments, in which NMs are sequentially exposed to artificial digestive fluids, aimed at understanding how their physicochemical properties change during digestion and how these changes affect their uptake and toxicity.
The authors found a large amount of high-quality data but also identified several knowledge gaps, particularly in the characterisation and standardisation of co-culture models. The physiological relevance of Caco-2 cells for NM translocation may be improved by incorporating several cell types in co-culture models, such as cells that facilitate the conversion of Caco-2 into M cells, which are involved in particulate uptake, or mucus-secreting cells. However, the conversion of Caco-2 cells into M cells has rarely been confirmed, with only 6 of the 24 studies reviewed providing evidence of their presence. Additionally, the exposure times were often too short (less than 24 hours) to be relevant for NM uptake, and the doses studied were often not comparable to typical daily human intake levels.
Despite these limitations, the authors state that the robustness and relevance of in vitro intestinal models are continually improving, making these approaches increasingly valuable, especially with enhanced interlaboratory reproducibility and regulatory acceptance.

Reflections by RIVM

To align with the 3R principles  (Replacement, Reduction an Refinement of animal experimentation) and improve the efficiency of NM risk assessment, the need for NAMs has been recognised in a joint report by the European Chemicals Agency (ECHA) and the European Union Observatory for Nanomaterials (EUON). The review offers valuable insights into the state of the art of NAMs for the intestinal fate of NMs. There is sufficient experimental data to understand the strengths and limitations of each model, allowing progress from the development phase to the validation phase.
Currently, the OECD is working on a guidance document addressing the intestinal fate of NMs. The draft guidance aims to harmonise and strengthen the scientific and regulatory foundations for assessing the intestinal fate of ingested NMs. The document provides recommendations for selecting appropriate methods and outlines important considerations for assessments. Furthermore, in agreement with the review article, the OECD emphasises the need for standardised operating procedures (SOPs) and provides exemplary SOPs for the proposed experimental procedures. Constructing SOPs and standardised reporting formats that adhere to the FAIR (findable, accessible, interoperable and reproducible) principles is a crucial step towards achieving standardisation and transferability of results.
The draft guidance has been shared for comments and will undergo a second round of public consultation. The guidance document, along with the exemplary SOPs, represents a first step toward establishing a test guideline. OECD test guidelines are recognised internationally and can support chemical and NM safety regulations in many countries, making them an essential part of performing a NAM-based risk assessment.
In conclusion, the strengths and limitations of NAMs in addressing the intestinal fate of NMs are increasingly well understood, and important steps towards harmonisation are being taken. However, before regulatory acceptance, the predictive performance of these methods in human scenarios must be assessed. Additionally, a better understanding of how the results can support NM safety assessment and other regulatory questions related to NM safety is needed.