The lung surfactant bioassay for hazard identification of airborne compounds: Principles, procedures and potential regulatory use

Emilie Da Silva*

*Corresponding author for this work

Research output: Book/ReportPh.D. thesisResearch

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Abstract

To protect workers and consumers, requirements for the hazard identification of inhaled chemicals and particles are set under a number of regulatory schemes, such as the regulation for the Registration, Evaluation, Authorisation and Restriction of Chemicals. While toxicity testing has traditionally been based on animal testing, there are scientific, ethical, and regulatory incentives to move away from in vivo methods. The development of alternative methods to replace experimental animals has been the focus of much investigation and it was stated in the European chemical strategy for 2020 that “safety testing and chemical risk assessment need to innovate in order to reduce dependency on animal testing but also to improve the quality, efficiency and speed of chemical hazard and risk assessments”. For hazard identification of airborne compounds, an alternative method to the existing test guidelines in animals has yet to be developed and accepted for regulatory use.
Unlike most studies focusing on in vitro cell models, the lung surfactant bioassay presented in this thesis is a cell-free system. The method is based on the study of lung surfactant, a complex mixture of lipids and proteins. One of the lung surfactant’s critical roles is to ensure effortless breathing by decreasing the surface tension at the air-liquid interface in the alveoli. The layer of lung surfactant is the first entity which inhaled chemicals and particles will encounter in the deepest part of the lungs. The lung surfactant coating the inside of the alveoli was identified as a toxicological target of inhaled compounds.
In this context, the overall objective of this thesis was to further develop and characterise the lung surfactant bioassay as well as to investigate its adequacy and regulatory readiness for hazard identification of airborne compounds. The specific aims were (i) to investigate the mechanism of toxicity of inhaled compounds for the lung surfactant function at the molecular level, (ii) to characterise the lung surfactant bioassay, (iii) to evaluate the suitability of the method to predict adverse lung effects in humans and in rodents, and finally (iv) to discuss the intended purpose and the regulatory readiness of the lung surfactant bioassay.
Empirical evidence is provided that shows the mechanistic relationship between the interaction of airborne chemicals with lung surfactant (molecular initiating event) and lung surfactant function inhibition (key event 1). In addition, the series of key events initiated by the interaction of chemicals and particles with lung surfactant and leading to immediate adverse lung effects is presented. Using these observations as a foundation, the principles and procedures of the lung surfactant bioassay are described. This thesis reports the use of the method with a large range of test compounds of relevance to workers and consumers and under different modes of exposure. A number of methodological considerations related to the test system and the testing conditions for studying lung surfactant function inhibition by airborne compounds are identified. These parameters can influence the measurements of surface activity of the lung surfactant before and during exposure to a test compound.
The results of this thesis indicate that the bioassay is suitable to predict adverse lung effects in vivo occurring immediately after inhalation of airborne compounds. Inhibition of the lung surfactant function in vitro correlated well with the decrease in tidal volume following exposure to bile salts, zinc oxide nanoparticles, impregnation spray products, and with respiratory clinical signs of toxicity occurring up to two hours post-exposure in rodents exposed to fragrance materials and industrial chemicals. Lung surfactant function inhibition was also observed for test compounds leading to adverse lung effects after human exposure. A number of weaknesses of the method were identified, the most important one being the estimation of inhibitory doses.
The lung surfactant bioassay appears to be a valuable tool for assessing the potential for immediate adverse lung effects of airborne compounds. This is based on the mechanistic relevance of the method, the correlation with animal studies and few human case reports, and the closeness of the results within and between experiments. This thesis argues that, already at this stage, the method is ready for application in industry. Specifically, the lung surfactant bioassay can be used in the context of hazard screening and prioritisation of potential compounds of interest to move forward in development. The findings of this work will be of importance to the scientific community and industries for the production of chemicals and products with the potential to be inhaled.
Original languageEnglish
Place of PublicationKgs. Lyngby
PublisherTechnical University of Denmark
Number of pages55
Publication statusPublished - 2021

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