Technical report 122

In vitro methods

The successful development of in vitro assays with cultured lung cells and aerosols have been critical for fostering predictive screening tests during the early phases of product development and mitigate some acute inhalation toxicity testing efforts. The potential advantages of these tests result from simpler, faster, and less expensive acute studies when compared to their in vivo counterparts. Perhaps more importantly, accurate and reliable development of in vitro testing systems will result in the reduction of animal use (3Rs) for screening studies of potential pulmonary test materials. Currently however, the accuracy of predictive results from in vitro screening studies for estimating in vivo pulmonary effects of respirable particles is unreliable, although there are a number of initiatives underway to develop such reliable testing schemes.

Although some progress has been made in the development of alternative (in vitro) methods to estimate in vivo toxicity endpoints of substances, reliable and predictive methodologies are still lacking. Most approaches thus far have revealed a lack of convergence when comparing particle induced effects when comparing in vitro vs. in vivo results on identical or similar particle-types (Seagrave et al, 2005, Sayes et al, 2007). These studies have utilised inflammatory and cytotoxic endpoints and reported little correlation among the biomarkers (e.g., inflammation/cytokines and LDH indices (Sayes et al, 2007).

The lung is a complex organ system comprised of many different interdependent cell-types (i.e., types I and II alveolar epithelial cells, macrophages, interstitial cells, vascular cells, etc.), thus the pulmonary microenvironment and the complex interactions in the lung which occur following inhalation and subsequent deposition of particles is difficult to simulate using in vitro techniques. Numerous studies have reported toxicity results with single cell-types. However, these are rather simplistic in scope and ignore the complex interactions in the lung milieu that occur following exposure to aerosols of particles, bacteria, etc.

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Ng and Warheit (2013) have attempted to develop a complex in vitro system which can, in part, simulate particle phagocytosis, an important aspect in the simulation of lung defence responses to particles, using lung epithelial – macrophage cellular co-cultures. The development of such in vitro techniques may serve to expedite the transition from the current animal-based testing system to one that is based primarily on human cell lines and in vitro assays. Such reproducible, accurate, and validated cell-based in vitro assays for assessing pulmonary hazards will have important benefits in screening more compounds in a faster, more reliable, and less expensive manner. In earlier comparative reports, Warheit et al (2007, 2009a,b) published the results of three studies demonstrating that in vitro pulmonary toxicity studies with particulates in a submerged culture system did not correlate with the measured in vivo effects when assessing identical particulate-types. Therefore, it would be useful to develop and implement a more efficacious/predictive cell culture/exposure system, in order to bridge the major technical gap on simulating in vitro exposures with relevance to the in vivo lung micro-environment. During the past year, some progress has been made in transitioning (and optimising) from a submerged in vitro cell culture system to a more physiologically relevant, in vitro air-liquid interface (ALI) system, using Transwell® Permeable Support devices (microporous membranes), for providing co-cultures (of rat lung epithelial L2 cells and NR8383 alveolar macrophages) applicable to potential in vitro cell exposures and toxicity assays. Certain cell growth conditions and parameters, and the applicability of several toxicity (XTT, LDH, and cytokine release) assays to the ALI co-culture cell model, are being confirmed and verified. Since an advanced aerosol exposure system is critical for exposing the co-cultures on ALI, Warheit reports that they are now testing a new device, the Nano-Aerosol Chamber for In vitro Toxicology (NACIVT); while developing methodologies to generate and document aerosol exposure concentrations at verifiable dose metrics. The output of this system will be to measure a variety of cellular toxicity end-points (of the co-cultures), namely, cytotoxicity indices, as well as generation of several inflammatory cytokines in a dose-dependent, time-course experimental protocol. The efficacy and validation of this approach remains to be determined. The proposed studies are designed to provide greater sophistication and relevance to the in vitro ALI cell exposure system by assessing the performance of the NACIVT to provide informative and reproducible results. Collectively, these efforts could expedite and facilitate the inevitable transition (as promulgated in the National Academy of Sciences Toxicology in the 21st Century report) from the current animal-based testing system to one that is based, in part, on more sophisticated in vitro toxicity and screening tests.

To summarise this Section, validation of the predictive value of in vitro screening assays for gauging in vivo pulmonary toxicity of particulates will be essential. In addition, study designs will require a focus on developing test with relevant particle dosimetry and realistic doses, and time course studies to gauge the sustainability of any measured endpoints. The cell-types used in the in vitro study designs should simulate respiratory tract point-of-entry routes of exposures. These integrated components are important prerequisites for developing in vivo predictability responses.