InVitro Toxicity Testing of Air Contaminants

The study of the toxic effects of inhaled chemicals is typically more challenging due to the technology required to generate and characterize the test atmospheres, and to develop effective and reproducible techniques for the exposure of cell cultures to airborne contaminants. The generation and characterization of known concentrations of air contaminants and reproducible exposure conditions requires different equipment and techniques (Fig. 4.1). For example, inhalation exposure systems involve several efficient and precise subsystems, including: a conditioned air supply system; a suitable gas or aerosol generator for the test chemical; an


Sampling & analysis


Exhaust system

Fig. 4.1 The components of a test atmosphere generation system (adopted from [62]).

atmosphere dilution and delivery system; an exposure chamber; a real-time monitoring, sampling and analytical system; a filter or scrubbing system; and an exhaust system [15, 60, 61].

A practical approach for in-vitro respiratory toxicity testing has been proposed by the European Centre for the Validation of Alternative Methods (ECVAM) [17]. This systematic approach is initiated with the consultation of existing literature, evaluating the physico-chemical characteristics of test chemicals, and predicting potential toxic effects based on SARs. The physico-chemical characteristics of chemicals such as molecular structure, solubility, vapor pressure, pH sensitivity, electrophilicity and chemical reactivity are important properties that may provide critical information for hazard identification and toxicity prediction [24, 34].

Initial in-vitro tests should be conducted to identify likely target cells and the toxic potency of test chemicals. Based on the obtained result, in-vitro tests may be followed by a second phase using the following cells: nasal olfactory cells, airway epithelial cells, type II cells, alveolar macrophages, vascular endothelial cells, fibroblasts, and mesothelial cells [17]. Moreover, the advantages and limitations of using different cell types and endpoints for toxicity measurement of air contaminants have been discussed [17]. Whilst over ten main cell types have been identified in the epithelium of the respiratory tract, for the assessment of respiratory toxicity it is important to utilize specific cell types with appropriate metabolizing activities. Therefore, freshly isolated cells maintained in suitable culture media that can mimic biotransformation activities and cellular functions comparable to the in-vivo environment are preferred to long-term cultures or cell lines that may differentiate, lose their organ-specific functions, and lack the enzyme systems required for biotransformation [17, 28]. It has been suggested that the endpoints used should be selected based on knowledge of the toxic effects of test

Exposure chamber

Table 4.3 Indirect and direct in-vitro exposure techniques developed for the study of the toxicity of air contaminants.

Exposure technique

Exposure achievement procedure

Indirect methods

Exposure to the test chemical itself

Exposure to collected air samples

Cells are exposed to test chemicals solubilized or suspended in culture media.

Cells are exposed to air samples collected by filtration or impingement methods.

Direct methods

Submerged exposure condition

Intermittent exposure

Continuous direct exposure at the air-liquid interface

Test gas is introduced to cell suspension under submerged conditions using impinger or vacuum test tubes.

Cells are periodically exposed to gaseous compound and culture medium at regular intervals using variation of techniques: rocker platforms, rolling bottles.

Cells are continuously exposed to airborne contaminants during the exposure time, usually on their apical side, while being nourished from their basolateral side using; collagen-coated or porous membranes permeable to culture media.

chemicals, and should always include cell viability testing in at least two different cell types. A better understanding of mechanisms involved in respiratory toxicity, as well as the development of standardized and reproducible in-vitro exposure and delivery systems which simulate inhalation exposure in vivo, were encouraged by the ECVAM workshop [17].

To evaluate the potential applications of in-vitro methods for studying respiratory toxicity, more recent models developed for the toxicity testing of airborne contaminants have been reviewed [62, 63]. The toxic effects of air contaminants have been studied using several indirect and direct in-vitro exposure techniques (Table 4.3).

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