The effects of exposure are investigated in multiple ways, mainly using volunteers, laboratory animals or cultured (lung) cells. RIVM examines how the dose, or amount of a substance, influences both the nature and severity of effects. Additionally, we explore how such effects depend on the pattern of exposure. For example, we study what happens after brief exposure to a high, ‘peak’ dose versus a prolonged low dose. Furthermore, our research examines whether effects observed in toxicological studies using laboratory animal or cell models are also evident in epidemiological studies of human populations. As the effects of inhalation are often not confined to the lungs, we investigate potential effects on other organs such as the heart, brain and kidneys as well.

Test atmosphere

There are various methods available for dispersal of substances into the air in order to investigate their toxicity when inhaled. For example, this can be achieved by evaporating volatile substances or by nebulising liquids or particles such as fine particulate matter, nanomaterials and microplastics. These processes create what is known as a test atmosphere that contains gases, vapours, liquid or solid aerosols, or a mixture of these components.
Further research can then be conducted on, for example, the hazards of a test atmosphere or a substance’s spread through the body.

Physicochemical properties

• A substance’s potential to cause harm depends on its form, structure and composition. These are collectively referred to as its physicochemical properties. RIVM uses a variety of instruments and techniques to characterise substances and test atmospheres:
  Scanning Electron Microscopy (SEM); this technique visualises the 3D structure and composition of materials
• Transmission Electron Microscopy (TEM); TEM allows for viewing cross-sections at very high magnification, enabling the observation of internal structures within cells
• Particle size analysers; these instruments determine the size distribution of aerosols or particles present in the air or in a liquid
• Condensation particle counters; these instruments measure the number of particles in the air
• Offline analysis; collection of samples for offline analysis using techniques such as mass spectrometry

ALI systems: Nebulisation and continuous flow

RIVM uses various methods to expose cells:

1. Direct liquid application; this involves dispersal of a test substance in a liquid for direct application to cultured cells. The advantage of this method is that it requires only a small quantity of a substance. However, it is not suitable for volatile substances. In addition, it exposes cells to the full amount all at once, unlike the gradual exposure that occurs when a substance is inhaled in reality.
2. Nebulisation; a test substance is nebulised above the cells, creating a cloud that settles on top of them. The advantage of this method is that it is simple and requires a relatively small quantity of the substance. Nebulisation also more closely resembles how lung cells are exposed to a finely dispersed substance in practice. One disadvantage may be that cells must be exposed to the substance in a liquid. Furthermore, this method is not suitable for volatile substances and the cells are exposed to the full amount of a substance in less than 2 minutes, a relatively short timespan.
3. Continuous flow exposure; this involves a continuous flow of a test atmosphere over the cells, closely mimicking actual inhalation exposure. This is a method suitable for both volatile substances and gases. Disadvantages are the complexity involved in creating and measuring an appropriate test atmosphere and the relatively large quantities of test substance required.

Nose-only and whole body

There are two methods for exposing laboratory animals to a test atmosphere:

1. Whole Body systems, which expose the entire animal
2. Nose-only systems, which expose only the animal’s nose

Collection of fine particulate matter

RIVM uses several methods to collect fine particulate matter. In all cases, air is drawn in and particles present in the air are captured using a suitable substrate such as a filter.

• A High Volume Cascade Impactor draws in air at a rate of 900 L/min. It collects fine particulate matter into bins in three size ranges: coarse (2.5-10 µm), fine (0.1-2.5 µm), and ultrafine (<0.1 µm). The picture below illustrates how this system is used to collect particles from brake wear for toxicological research.
• The Versatile Aerosol Concentration Enhancement System (VACES) draws in air which is saturated with water vapour and then cooled significantly. A thin layer of water encapsulates the particles, making them larger and easier to capture. Using devices known as virtual impactors, air is extracted from the aerosol until the remaining air contains up to 20 times as many particles, a process referred to as concentration. The particles can then be filtered out of the air or collected in a liquid. Alternatively, after dewatering, the air containing the particles may be used to expose cells, animals, or humans directly.
• The Harvard Impactor draws in 10 L/min of air, collecting any particulate matter present with an aerodynamic diameter smaller than 10 or 2.5 µm onto a filter.

Mobile laboratories

Exposure studies can also be conducted on-site. The biggest advantage of this approach is that exposure occurs directly at the location of the emissions. This eliminates the need to collect substances, transport them elsewhere, and then disperse them into the air again, as each of these steps can alter the physicochemical properties of a test substance. This is particularly relevant in case of complex mixtures such as diesel emissions or oil fumes. RIVM operates two climate-controlled mobile laboratories for on-site exposure research.

Computer models and exposure models

Besides conducting lab experiments, RIVM also uses computer models to perform calculations. Such models can be useful in estimating the amounts of a substance deposited in the lungs due to inhalation and in determining how substances spread through the body. Two important types of models:

• Physiologically Based Kinetic (PBK) models; these models simulate how a substance spreads through the body.
• Multiple-Path Particle Dosimetry (MPPD) models; a computer application that includes deposition models of the airways. It simulates the deposition and clearance of inhaled particles in the lungs of humans and laboratory animals. The MPPD tool is used in risk analysis and research to help predict where particles may deposit within the respiratory system, such as in the nose, bronchi or alveoli. The model can aid the design of studies using ALI systems to expose cells to aerosols.

Originally developed in collaboration with and on behalf of RIVM, the MPPD model is currently managed by Applied Research Associates and the US Environmental Protection Agency.

More information

• MPPD: Multiple-Path Particle Dosimetry Model