METHODS IN INHALATION TOXICOLOGY Edited by Robert F. Phalen, Ph.D. Boca Raton London New York CRC Press is an imprint of the Taylor & Francis Group, an informa business CRC Press METHODS IN THELIFESCIENCES GeraldD. Fasman - Advisory Editor Brandeis University Series Overview MethodsinBiochemistry JohnHershey Departmentof BiologicalChemistry Universityof California CellularandMolecularNeuropharmacology Joan M. Lakoski Department of Pharmacology Penn State University Research MethodsforInbredLaboratoryMice JohnP.Sundberg The Jackson Laboratory BarHarbor, Maine MethodsinNeuroscience JosephM. Corless SidneyA. Simon DepartmentofCell Biology, Department ofNeurobiology Neurobiology and Ophthalmology Duke University Duke University MethodsinPharmacology JohnH. McNeill Professor andDean FacultyofPharmaceutical Science The University ofBritish Columbia MethodsinSignal Transduction JosephEichbergyJr. Department of Biochemical and Biophysical Sciences Universityof Houston Methodsin Toxicology EdwardJ.Massaro Senior Research Scientist National Health and EnvironmentalEffects Research Laboratory Research Triangle Park, North Carolina METHODS IN TOXICOLOGY EdwardJ.Massaro -Advisory Editor Senior ResearchScientist NationalHealthandEnvironmental EffectsResearch Laboratory Reasearch Triangle Park,North Carolina The CRC PressMethods in Toxicology Series provides the reader with a step-by-step approach to each ofthe up-to-date methods and techniques which arepresentedinaclear andconciseformat. Topics coveringallaspects ofthe methodsthat impacton toxicologyarebeing reviewed for publication. Published Titles Methods inRenal Toxicology,Rudolfs K. Zalups and Lawrence H. Lash Molecularand CellularMethods in Developmental Toxicology, George P.Daston Methods in Inhalation Toxicology,Robert F.Phalen Forthcoming Titles PCR Application toMolecular Toxicology, JackVanden Heuvel CRCPress Taylor & Francis Group 6000 B roken Sound Parkway N W, Suite 300 Boca R aton, FL 33487-2742 First i ssued in h ardback 2 019 © 1 997 by T aylor & Francis Group, LLC CRC Press is an imprint ofTaylor & Francis Group, an Infonna b usiness No claim to original U .S. Government works ISBN-13: 978-0-8493-3343-9 (Pbk) ISBN-13: 978-1-138-47368-3 (hbk) This book contains information obtained from authentic and highly regarded sources. Reasonable efforts h ave b een m ade t o p ublish r eliable d ata a nd i nformation, b ut t he a uthor a nd p ublisher c annot assume r esponsibility for t he validity of all materials o r the consequences of their use. T he authors and publishers h ave a ttempted t o trace the copyright holders o f all material reproduced i n this publication and apologize to copyright holders if permission t o publish in this form has n ot been obtained. I f any copyright m aterial h as n ot b een a cknowledged please w rite a nd l et us k now so w e may rectify i n a ny future r eprint. Except as permitted u nder U .S. Copyright Law, no part o f t his book may be reprinted, reproduced, transmitted, or u tilized in any form by any electronic, mechanical, or o ther m eans, now known or hereafter i nvented, i ncluding p hotocopying, m icrofilming, a nd r ecording, o r i n a ny i nformation s tor­ age o r r etrieval s ystem, w ithout w ritten p ermission f rom t he p ublishers. For permission t o p hotocopy o r u se m aterial e lectronically f rom this w ork, please a ccess www.copy­ right.com (http://www.copyright.coml) or c ontact t he C opyright C learance C enter, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 0 1923, 978-750-8400. CCC is a not-for-profit organization that p ro­ vides licenses and registration for a v ariety of users. F or organizations t hat have b een granted a p hoto­ copy license b y t he CCC, a s eparate s ystem o f payment has b een arranged. Trademark Notice: P roduct o r corporate names may be t rademarks o r registered trademarks, and are used o nly f or identification a nd e xplanation w ithout i ntent t o i nfringe. Visit the Taylor &. Frands Web site at http://www.tayIorandfrancis.com and the eRC Press Web site at http://www.crcpress.com Cover design: Dawn Boyd Library of C ongress Card Number 9 6-25496 Library o f Congress Cataloging-in-Publication Data Methods in inhalation toxicology I edited by Rober! F. Phalen. p. cm. --(CRC Press methods in toxicology) Includes bibliographical references and index. ISBN 0-8493-3343-1 1. Pulmonary toxicology--Animal models. 2. Air--Pollution­ -Toxicity testing. I. P halen, Rober! F., 1940- . H. Series. [DNLM: I. R espiratory System--drug effects. 2. Toxicology­ -methods. 3. Air P olIutants--toxicity. WF 100 M592 1996] RCnO.M48 1996 615.9'02--dc20 DNLMlDLC for Library of C ongress 96-25496 CIP Abstract Today, and in the foreseeable future, an appropriate understanding of the biomedical effects of potentially significant natural and anthropogenic air contaminants will require conducting inhalation exposure studies employing whole living subjects. Conducting such studies is aspecializedresearch activ­ ity that draws upon skills from avariety of scientific disciplines. How knowl­ edge from these disciplines is integrated into the design and conduct of inhalation studies is the subject of this book, which was written by an expe­ rienced, active team of research scientists. Each of the vital aspects germane to the conduct of such studies is discussed in detail, including the selection of animal subjects and the related quality-control and ethical considerations, airpurification methodology, exposure atmosphere generation and character­ ization, inhalationexposure systems, andreal-time andpost-exposure biolog­ ical assessments. The book, which is targeted to students in toxicology programs andscientists contemplatingperforminginhalation studies,includes comprehensive lists andaddresses of suppliers ofuseful equipmentandmate­ rials relevant to each of the major aspects involved with the performance of valid, reproducible inhalation toxicology studies. Preface Welive and breathe immersedina seaofair.Inaddition to themajor gaseous components, oxygen and nitrogen, countless additional gases, vapors, and particles are always present in the atmosphere and are available to deposit in the respiratory system. Some ofthese contaminants arenatural, andsome are anthropogenic. Of the anthropogenic substances, some arebeneficial (such as aerosolized medications), and some are harmless or harmful depending upon their concentrations. Ifwe are to maximize human wellbeing, we must study andunderstand thebiomedicaleffects ofeachofthe potentially significantair contaminants.Today, andinthe foreseeable future, this study andunderstand­ ing will require conducting inhalation studies using whole living subjects. In some studies, the subjects will be human volunteers, but in most studies laboratory animals will be involved. Conducting such studies is a specialized research activity that draws upon skills taken from numerous scientific disci­ plines. These disciplines include chemistry, physics, engineering, biology, physiology, microbiology, medicine, pathology, and others as well. How knowledge from these areas is integrated into the design and conduct of inhalation studies is the subject of this volume. Thechapters in thisbookwerewritten byanactiveteamofscientistswho have worked together in a modem inhalation toxicology program at the Air Pollution Health Effects Laboratory at the University of California, Irvine. Performing such studies has been amajor activity ofthis team for the past 10 years. In addition, these authors have trained students in research methods, and they have established and taught several formal courses related to the topics in this book. In short, each author is writing about his current daily activities and within the scope of his formal training and experience. It is hoped that this volume will provide a solid foundation for the readers — a foundation that will support further specialized learning and will lead to success in conducting theirown inhalation studies. In addition to the authors, several people have contributed significantly to the material herein. The chapters were reviewed and improved by the technical staffof the Air Pollution Health Effects Laboratory. Special thanks are due to Richard C. Mannix, M.S.; Charles Bufalino, M.S.; LauraA. Hoff­ man, B.A.; and Michael J. Oldham, M.S. Additional editing, checking, for­ matting, and word-processing were performed by Marie C. Tonini. Finally, this bookis dedicatedwith gratitude andrespectto the research subjects; they have been part of the team as well. RobertF.Phalen, PhD. Irvine, CA The Editor Robert Franklynn Phalen, Ph.D., directs the Air Pollution Health Effects Laboratory at the University of California, Irvine (UCI). He also holds two academic appointments in the College of Medicine at UCI: Professor in the Department ofCommunity andEnvironmental Medicine andProfessorin the Center for Occupational and Environmental Health (in the Department of Medicine). He is amember of the faculty for the Graduate Program in Envi­ ronmental Toxicology at UCI, and he teaches medical students, medical res­ idents, graduate students, and undergraduate students in courses at several universities including UCI, UCLA, and USC. Dr. Phalen graduated in 1964 from San Diego State University with a B.S. in physics (and a mathematics minor) and from the same institution in 1966 with an M.S. degree in physics with a specialty in nuclear physics. In 1971, he obtained a Ph.D. in biophysics, with specialization in inhalation toxicology, from the University of Rochester (Rochester, NY). His postdoc- torial research inaerosol physicsand inhaledparticledepositionmodelingwas conductedatthe Inhalation ToxicologyResearch InstituteinAlburquerque, NM. In 1972, Dr. Phalenjoined the College of Medicine at UCI to establish the Air Pollution Health Effects Laboratory, which has since conducted over 100 studies relating to the toxicology of air pollutants. These studies have been supported by avariety of federal agencies (including the U.S. EPA; the National Heart, Lung and Blood Institute; the National Institute of Environ­ mental Health Sciences; andtheCenters forDisease Control andPrevention), California state agencies (California Air Resources Board,the Department of Health Services, and others), and numerous private agencies (including the Electric PowerResearch Institute, the Health Effects Institute, the Charles C. Stocking Trust Fund, Southern California Edison Co., the Nickel Producers Environmental Research Association, and others). His research has covered several topics, including respiratory tract defenses, comparative respiratory tract anatomy, basic aerosol science, inhaled aerosol deposition models, par­ ticle and gas toxicity, andthe inhalability of large particles.