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Operating Procedures to Improve Efficiencies of In vitro Exposure Systems at the Air-Liquid Interface
Citation:
Higuchi, M. AND J. Zavala-Mendez. Operating Procedures to Improve Efficiencies of In vitro Exposure Systems at the Air-Liquid Interface. Society of Toxicology Annual Meeting, San Antonio, TX, March 11 - 15, 2018.
Impact/Purpose:
The expanding use of in vitro exposure systems for toxicity assessments has created regulatory concerns. Many of these same concerns surround the proper conduct of in vivo inhalation toxicology studies that are addressed in guidelines and Good Laboratory Practice (GLPs) regulations. We acknowledged that this area (exposure techniques) is in rapid development, and there is limited experience now on best practices to describe new in vitro exposure methods.
Description:
The expanding use of in vitro exposure systems for toxicity assessments has created regulatory concerns. Many of these same concerns surround the proper conduct of in vivo inhalation toxicology studies that are addressed in guidelines and Good Laboratory Practice (GLPs) regulations. We acknowledged that this area (exposure techniques) is in rapid development, and there is limited experience now on best practices to describe new in vitro exposure methods. For example, OECD GLP No 14 - The Application of the Principles of GLP to in vitro studies states “routine requirements for apparatus used in a GLP compliant environment apply equally to apparatus used for in vitro studies∙∙∙”. Unfortunately, the supplier/developer of in vitro exposure systems cannot know all potential applications of the specific exposure system by an end-user. Therefore, the in vitro method end-users must make the determination if the specific in vitro exposure system will meet the requirements of their experimental design. OECD GLP No. 14 document provides guidance for in vitro methods for regulatory use in human safety assessment, since most end-users will be GLP compliant test facilities. Non-GLP in vitro method users can profit from the use of the OECD document on Good In Vitro Method Practices (GIVIMP) guidance. In the specific cases when existing regulations and responsibilities are not defined. Previously, we compared various in vitro exposure systems for their ability to expose cells to particles and gases. Various factors, such as air flow rate, relative humidity, and temperature can adversely affect performance and reliability. For example, increasing the air flow rate in diffusion (gases & vapors) exposure systems, when air flow is perpendicular to the cells, increases gas delivery to wells via turbulence. To demonstrate this effect biologically, we used EPA’s cell culture exposure system (CCES) to expose BEAS-2B cells to air only (sham) controls. The experiments were aimed at “defeating the technology” of the CCES by determining the cells viability in a sham exposure. The controllable parameters of the exposures were total airflow rate, relative humidity, and temperature. Determining cytotoxicity via the CellTiter Glo assay using the worst set of operating parameters (low air flow rate and relative humidity) indicated an average cell viability versus incubator controls of 62%. These results show that air flow rate and relative humidity are critical factors that influences the biological effects. Characterization of in vitro exposure systems is needed prior to their use to understand their advantages and limitations for regulatory assessments. [Abstract does not necessarily reflect the views or policies of the U.S. EPA.]