Researchers have developed an Adverse Outcome Pathway (AOP) that describes how nanomaterials (NMs) can compromise the gut’s protective barrier, leading to a “leaky gut”. The AOP outlines the biological mechanisms through which ingested NMs can harm intestinal integrity and increase permeability. Despite existing uncertainties and knowledge gaps, the AOP and associated testing methods are relevant for the risk assessment of NMs.

An AOP for a compromised gut barrier by nanomaterials

Researchers have outlined a step-by-step process, known as an Adverse Outcome Pathway, describing how nanomaterials can potentially damage the gut’s protective barrier. They based the AOP on data from existing AOPs published in the AOP-Wiki and on considerations of biological plausibility. The researchers focused on NMs that can enter the body through food, but they can also be used in other industries.

What is an AOP?

The Organisation for Economic Co-operation and Development (OECD) defines the AOP as a description of a logical sequence of causally linked events (key events, KEs) at different levels of biological organization. An AOP outlines the sequence of connected events that lead to adverse health effects, starting from the exposure of a living organism to a potentially harmful stressor and continuing through to any resulting harm. AOPs are valuable tools for scientists, as they facilitate a more detailed and systematic assessment of risks and hazards, using modern methods that focus on understanding the underlying mechanisms involved. These methods are referred to as New Approach Methodologies (NAMs).

The risks of intestinal permeability

When certain NMs are ingested, they could harm the protective layer of the intestines, making it more permeable. This condition is often referred to as “leaky gut”. Individuals with a leaky gut can absorb more harmful substances from their diet, which increases their risk of becoming ill from chemicals found in food or the environment. Leaky gut can worsen various health issues, including inflammatory bowel disease and celiac disease.

The events leading to a leaky gut

The proposed AOP starts with a process called endocytosis, where cells take in substances from their surroundings. During this process, the cell membrane surrounds and engulfs the substances, forming a small bubble called a vesicle that carries the substances inside the cell for digestion. Once inside the cell, there are special compartments known as lysosomes that contain powerful digestive fluids designed to break down unwanted material. However, these digestive fluids can also be harmful if released into the cell, potentially leading to the death of intestinal epithelial cells and resulting in a more permeable (or leaky) intestinal wall.

The authors carefully reviewed existing studies and identified areas of uncertainty, inconsistencies in findings, and key gaps in knowledge. While the proposed mechanism makes sense from a biological perspective, there isn’t enough evidence regarding the specific NMs being discussed. This lack of evidence is largely due to differences in the properties of these materials, the biological systems they interact with, and the treatments and dose levels used in studies.

Reflections by RIVM

The authors have put forward an AOP for a relevant, potentially harmful effect related to ingested NMs. They carefully discuss both the strengths and weaknesses of the proposed AOP. A key highlight of the article is their suggestions of specific NAMs that can be used to test for specific KEs. However, they do not specify if these methods are specifically tailored to NMs. In certain cases, such as measuring cytotoxicity or membrane integrity, it is necessary to adapt these tests to account for the unique features of NMs, as reviewed in the NAMs4Nano project. Despite this limitation, the authors emphasised the importance of properly characterising NMs, in line with the European Food Safety Authority (EFSA) guidance (EFSA, 2011).

The authors note that part of the reason for the insufficient evidence is experimental variation. This points to the importance of having harmonised testing methods. For these methods to be accepted by regulatory bodies, they should not only be harmonised but also validated. Validation ensures that the testing methods are relevant, reliable, and reproducible across different laboratories.

Ideally, NAMs should be included in the standard test sets from organisations like the International Organization for Standardization (ISO) and OECD. However, achieving this requires extensive validation, coordination, prioritisation, and funding. In January a policy conference was organized to discuss with European policy advisors and decision makers how to accelerate the availability of regulatory accepted tests. More details and the chairman’s report can be found here. For NM-specific NAMs, there may need to be specific validation guidance to address their unique properties. Currently, the OECD is working on updating its Guidance Document on the validation and international acceptance of new or revised testing methods for hazard assessment.

Ideally, an AOP would apply universally to all chemicals and particles leading to a selected adverse outcome. According to RIVM, the proposed AOP is probably not relevant for conventional chemicals. For NMs, cell entry primarily occurs through endocytosis. However, for conventional chemicals, there are typically different mechanisms for cell uptake, meaning that the proposed AOP will not be initiated. While conventional chemical exposure can still harm the intestinal barrier, it will likely occur through a different mode of action, requiring a different AOP. 

Despite the uncertainties, knowledge gaps and need for validation and guidance, the use of AOPs and associated testing methods has the potential to be highly relevant for the risk assessment of NMs.