Simulated lung fluid can be used to simulate, in vitro, the process by which particulate matter enters the lungs and comes into contact with lung tissue fluid. It can be used to evaluate the proportion of target contaminants released from particulate chemicals emitted by consumer products into a lung-fluid-like environment. This method uses bioaccessibility as the core indicator. Compared with simply measuring the total concentration of contaminants, it is more suitable for reflecting the potentially accessible dose of target chemicals under inhalation exposure conditions and can be used for consumer product safety evaluation, particulate matter exposure research, and in vitro risk screening.

Keywords: simulated lung fluid; in vitro exposure simulation; particulate chemicals; consumer product safety; bioaccessibility; inhalation exposure; risk assessment; organic pollutants

1 Method Positioning and Evaluation Objectives

1.1 Respiratory Exposure Characteristics of Particulate Chemicals from Consumer Products

(1) Sources of particulate matter release

Consumer products may release particles carrying organic pollutants during use, friction, aging, breakage, or heating. After entering the human body through respiration, these particles may deposit in the respiratory tract or deep lung regions and come into contact with lung tissue fluid, alveolar surface fluid, or macrophage-associated fluid environments.

(2) Limitations of total concentration assessment

For hydrophobic or semi-volatile organic pollutants such as plasticizers, flame retardants, short-chain chlorinated paraffins, and polycyclic aromatic hydrocarbons, the total concentration does not equal the actual concentration accessible in the lungs. Some pollutants may be tightly bound to particle surfaces, polymer matrices, or carbonaceous particles and may be released only to a limited extent in lung fluid environments. Other pollutants may migrate relatively quickly into the liquid phase and therefore have the potential to further interact with cells or tissue barriers.

(3) Significance of release capacity assessment

Whether pollutants carried by particles can be released from the particle matrix is an important step in judging their potential health risk. The purpose of the simulated lung fluid in vitro exposure method is to evaluate this releasable fraction of pollutants in a lung tissue fluid-like environment, thereby providing a basis for bioaccessibility calculation and inhalation exposure risk assessment.

1.2 Role of Bioaccessibility in Risk Assessment

Bioaccessibility refers to the proportion of pollutants released from particles and transferred into body fluids or simulated body fluid environments. For particulate chemicals released from consumer products, bioaccessibility can be used to further convert “the total amount of pollutants in consumer products or particles” into “the potentially accessible fraction released under lung fluid conditions,” thereby reducing bias that may arise when risk judgment is based only on total concentration.

(1) Released phase

The released phase refers to the target pollutants transferred into the simulated lung fluid, representing the fraction that directly enters the liquid environment under lung tissue fluid-like conditions.

(2) Adsorbed phase

The adsorbed phase is usually captured by adsorption materials such as Tenax and is used to simulate continuous absorption or maintenance of a concentration gradient in vivo for migratable hydrophobic pollutants.

(3) Particle residual phase

The particle residual phase refers to pollutants that remain on the particles or carriers and have not been released. It can be used to assess the binding strength between the target compounds and the particle matrix and to support mass balance analysis.

2 Role of Simulated Lung Fluid in In Vitro Exposure Simulation

2.1 Core Simulation Medium

Simulated lung fluid is a type of research reagent used to simulate the lung tissue fluid environment in vitro. It provides stable and reproducible liquid-phase conditions for particle release experiments. In the evaluation of particulate chemicals from consumer products, simulated lung fluid is not used to reproduce the complete physiological system of the lung, but to establish a release model after particles come into contact with a lung-fluid-like environment, thereby analyzing the migration potential of target pollutants in lung-related fluid environments.

This type of product is suitable for evaluating the release, migration, and bioaccessibility of particulate chemicals under in vitro conditions and can be used together with filtration, extraction, adsorbed-phase capture, and instrumental analysis. This product is for research use only and is strictly prohibited for other uses.

2.2 In Vitro Simulation Logic

(1) Liquid-phase contact simulation

After particles enter the lungs, they come into contact with lung tissue fluid, alveolar surface fluid, or macrophage-associated fluid environments. Simulated lung fluid provides an experimental medium similar to lung body fluids, allowing contaminants in particles to undergo extraction, desorption, and migration in vitro.

(2) Pollutant release simulation

Target pollutants in particles may enter simulated lung fluid through surface desorption, pore diffusion, migration from the material matrix, and liquid-phase transfer. The degree of release is affected by the hydrophobicity of the target compound, particle size, material matrix, liquid-phase environment, incubation time, temperature, and other factors.

(3) Estimation of accessible dose

The core result of a simulated lung fluid experiment is not the total amount of the target compound, but the in vitro releasable fraction. This fraction can be used as an estimation parameter for lung-accessible dose, supporting risk classification of particulate chemicals from consumer products and prioritization for subsequent toxicity assessment.

3 Method Workflow

3.1 Particle Sample Preparation

Consumer product samples can be cut, abraded, cryogenically ground, sieved, or collected as particles according to realistic exposure scenarios. When comparing different samples, the particle size range, sample mass, processing method, and storage conditions should be standardized as much as possible, because particle size directly affects specific surface area, release rate, and contact efficiency with simulated lung fluid. Applicable samples include plastic products, rubber products, schoolbags, erasers, coating materials, textiles, children’s products, indoor settled dust, material wear particles, and aerosol sampling particles.

3.2 Simulated Lung Fluid Exposure Incubation

(1) Basic exposure system

Add a defined mass of particles into simulated lung fluid and incubate under dark shaking conditions at 37°C to allow sufficient contact between the particles and the simulated lung fluid environment. The incubation time can be set according to the release kinetics of the target pollutants. Short-term incubation is suitable for observing rapid desorption, while long-term incubation is suitable for evaluating slow release and matrix migration.

(2) Adsorption sink-coupled system

For hydrophobic organic pollutants, adsorption materials such as Tenax can be combined to construct an adsorption sink that continuously captures target compounds released from particles and migrating into the liquid phase. This design helps maintain a concentration gradient between the particles and the liquid phase and simulates the continuous removal of migratable components during in vivo absorption.

(3) Particle isolation system

When it is necessary to prevent particles from directly entering the adsorption material, isolation devices such as dialysis bags can be used to separate particles from the adsorbed phase. This design can reduce the risk of overestimating bioaccessibility caused by fine particles entering the adsorption material structure and being co-extracted.

3.3 Phase Separation and Pretreatment

(1) Simulated lung fluid released phase

After incubation, the simulated lung fluid can be filtered through a glass fiber filter membrane and treated by liquid-liquid extraction, solid-phase extraction, or other enrichment methods according to the properties of the target compounds. This fraction is used to determine the amount of pollutants that have entered the lung-fluid-like environment.

(2) Adsorbed phase

Tenax or other adsorption materials are extracted with organic solvents, concentrated, and cleaned up before analysis. This fraction is used to determine the amount of target pollutants released from particles and further migrated and captured by the adsorbent.

(3) Particle residual phase

The filter membrane, particle residues, or related isolation carriers are dried, ultrasonically extracted, column-cleaned, and concentrated before analysis. This fraction is used to determine the amount of pollutants that have not been released and remain on the particles.

3.4 Instrumental Analysis

Target pollutants can be analyzed using GC-MS, GC-MS/MS, LC-MS, LC-MS/MS, ICP-MS, ICP-OES, or other platforms according to their chemical properties. Volatile or semi-volatile organic pollutants such as PAEs, PAHs, and SCCPs are commonly analyzed using GC-MS-based methods. Compounds with higher polarity or poor thermal stability can be analyzed using LC-MS-based methods. Metal elements can be detected using ICP-MS or ICP-OES. During method development, internal standard recovery, procedural blanks, spiked recovery, and mass balance should be used to assess result reliability.

4 Bioaccessibility Calculation and Data Interpretation

4.1 Calculation Method

When the simulated lung fluid phase, adsorbed phase, and particle residual phase are all measured in the experimental system, the simulated lung fluid phase and adsorbed phase can be regarded as the accessible fraction, while the particle residual phase can be regarded as the unreleased fraction. The bioaccessibility fraction can be calculated as follows:

Bioaccessibility fraction (%) = (amount in simulated lung fluid phase + amount in adsorbed phase) / (amount in simulated lung fluid phase + amount in adsorbed phase + amount in particle residual phase) × 100%

In this equation, the amount in the simulated lung fluid phase reflects the fraction of the target compound directly released into the lung tissue fluid-like environment; the amount in the adsorbed phase reflects the fraction that can continue to migrate after release and be captured by the adsorbent; and the amount in the particle residual phase reflects the fraction that remains bound to particles or unreleased.

4.2 Data Interpretation

(1) High bioaccessibility

The target pollutant is more readily released in the simulated lung fluid environment, suggesting a higher potential for lung accessibility. Such samples should be prioritized for cytotoxicity testing, pulmonary epithelial barrier transport studies, or further exposure risk research.

(2) Low bioaccessibility

The target pollutant mainly remains in the particles, with limited short-term release capacity. In this case, actual lung exposure risk should not be inferred from total concentration alone, but should be evaluated together with particle retention time, long-term exposure frequency, and the possibility of chronic release.

(3) Increased proportion in the adsorbed phase

After release, the target compound can be continuously captured by the adsorption sink, indicating strong migration potential in the simulated lung fluid system. For hydrophobic organic pollutants, adsorbed-phase data help explain release behavior under sustained concentration gradient conditions.

(4) Abnormal mass balance

If the total recovery across the three phases is significantly low, possible causes such as volatilization loss, photodegradation, vessel adsorption, membrane material adsorption, extraction loss, cleanup loss, or instrumental response deviation should be investigated. Mass balance is the basis for reliable bioaccessibility results.

Table 1 Key evaluation steps in simulated lung fluid in vitro exposure simulation

5 Applications in Risk Assessment

5.1 Consumer Product Safety Screening

The simulated lung fluid method is suitable for safety screening of consumer products that may release inhalable or depositable particles. For plastic products, rubber products, children’s products, stationery, textile materials, and coating materials, the total concentration of target pollutants can first be measured, followed by simulated lung fluid experiments to evaluate the in vitro releasable fraction. This helps distinguish samples with high total content but low release risk from samples with both high total content and high release risk.

5.2 Comparison of Particle Sources

Particles from different sources vary in material matrix, carbonaceous components, mineral composition, particle size distribution, and surface properties, which can significantly affect pollutant release behavior. Simulated lung fluid systems can be used to compare lung-accessible release differences among different consumer products, different material batches, or different processing conditions, providing a basis for material safety assessment and pollutant migration mechanism analysis.

5.3 Risk Classification Judgment

In the evaluation of particulate chemicals from consumer products, total concentration, bioaccessibility, and toxicity data can be used together. Total concentration is used to assess pollutant loading level, bioaccessibility is used to evaluate release potential in the lung liquid phase, and cellular or in vivo experiments are used to further verify biological effects. This tiered strategy can improve the accuracy of risk assessment and the efficiency of experimental resource use.

6 Related Reagent and Material Selection

Table 2 Reagents for sample extraction, cleanup, and representative target pollutants in simulated lung fluid exposure assays

Table 3 Supporting materials for exposure system construction and quantitative analysis in particle-bound chemical bioaccessibility assessment

7 Usage Precautions

7.1 Research Use Only

Simulated lung fluid products are for research use only and are strictly prohibited for other uses. When particles, organic solvents, and potentially toxic pollutants are involved, experiments should be performed according to laboratory safety standards, and appropriate personal protection and waste liquid disposal methods should be selected according to the properties of the target pollutants.

7.2 Particle Size and Sample Homogeneity

Particle size affects specific surface area, release rate, and interpretation related to lung deposition. When comparing different samples, the particle size range and sample preparation conditions should be standardized. If particle size differs greatly, its possible influence should be separately discussed in the result analysis.

7.3 Background Contamination Control

PAEs, PAHs, and some flame retardants are easily affected by laboratory environment, plastic consumables, and solvent background. Glassware should be preferred during experiments, and simulated lung fluid blanks, reagent blanks, procedural blanks, and spiked recoveries should be included to ensure reliable analytical results.

7.4 Applicability Boundaries of Results

Simulated lung fluid experimental results reflect in vitro release potential and bioaccessibility, and are not equivalent to actual human absorption rate. Real absorption processes are also affected by the pulmonary epithelial barrier, macrophage phagocytosis, mucus clearance, metabolic transformation, and blood circulation. This method is suitable as an in vitro screening and tiered evaluation tool in risk assessment.

8 Frequently Asked Questions

8.1 Why is the simulated lung fluid method more suitable than total concentration measurement for evaluating inhalation exposure?

Total concentration only indicates how much target pollutant is present in particles, but does not indicate how much can be released after entering the lung fluid environment. The simulated lung fluid method can obtain the in vitro releasable fraction and is more suitable for inhalation exposure-related risk assessment.

8.2 Is bioaccessibility the same as bioavailability?

No. Bioaccessibility refers to the proportion of pollutants released from particles into simulated lung fluid or the adsorbed phase. Bioavailability also involves cellular uptake, transmembrane transport, metabolism, and entry into systemic circulation.

8.3 Why is it necessary to analyze the particle residual phase?

The particle residual phase is used to determine the proportion of pollutants that remain unreleased and helps establish mass balance. Without analyzing the residual phase, it may be impossible to determine whether a low-release result reflects true non-release or is caused by pretreatment loss or analytical error.

8.4 Which pollutants are suitable for evaluation using this method?

This method is applicable to short-chain chlorinated paraffins, phthalates, polycyclic aromatic hydrocarbons, flame retardants, and other organic pollutants in consumer product particles that can be analyzed using corresponding instrumental methods. Metal elements can also be incorporated depending on the detection platform and pretreatment method.

The simulated lung fluid in vitro exposure method evaluates the release proportion and bioaccessibility of particulate chemicals from consumer products by constructing a liquid-phase environment similar to lung tissue fluid. This method can further convert total pollutant concentration into lung-accessible release data, providing experimental support for consumer product inhalation exposure risk assessment, material safety screening, and research on pollutant migration from particulate matter.