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9出 Asia-PacificConferenceonCombustion， GyeongjuHilton，Gyeongju，Korea
19四 22May2013
Developmentofinvitrotoxicitymethodsforfirecombustionproductsby usinghumanA549lungcels
TadashiKonishi1，KimKwang圃 il2，KiHunNam2，ParkHyeJeong2，AkiraNarumi
10itaNationalCollegeofTechnology，Oita870目 0152，Japan 21njeUniversity，Gimhae621聞 749，Korea 3KanagawaInstituteofTechnology，Kanagawa243-0292，Japan
Abstract
Firegastoxicityisanesentialcomponentofanyfrehazard analysis.TheuseofhumancelA549，ahumanlungalveolar typeIcelline，invitrocytotoxici旬 0宜ersanalternative methodtolaboratoryanimals.Cytotoxicityandmorphological asesment of combustion by-products 企om four plastics: acrylonitrile-butadiene-styrenes(ABS)，nylons，Urethaneand Melarnine， have be eninvestigated.The human cel l exposure systemcanenableA549celtoaplythrediferentincubation gases，95%airwith5%CO2，100%airandthecombustiongas ThefolowingisHCNgenerationorderasmeasuredformthe mos中roductive to least productive: ABS>Urethane >Nylon measuredbygaschromatograph.Theexposureto95%airwith5% CO2，after the time periods of 24，48 and 72 h，no morphologicalchangewasobserved.WhileA549celswere exposedtotheeftluentsproduced企omABS，itapearedthat the cel ls began to shrink in size and cel ls were ir regular1y shapedandhadobscurecelboundaries.Thecelviabilityand live/deadcelcountshowedthatA549celsprogresed丘om84% viableattirne0.5htolesthan20%viableby4hours.The folowingtoxicityrankingasobserved企omthemosttoxicto theleastto氾c:ABS>Nylons>Urethane>Melarnine.
1 Introduction
Recently， Japanese residential houses become more insulatedand highly airtight for energy saving. Once a fire ocursinthosehouses，lowoxygenenviromnentcaneasilybe formed by incomplete combustion.As a result， residents are exposed to various toxic gases inc1uding carbon monoxide (CO)，hydrogencyanide(HCN)，aldehydes，hydrogenchloride， benzene， sulphur dioxide， dichlorofluoromethane， and particulates.Thesetoxicgasesaregeneratedfromtheacry1ate resinusedforoficeautomationequipment，ABSresinandthe po1yurethaneusedforbedc1othes，Japaneseflorcushion，or newsyntheticbuildingmaterials[l].Furthermore，frefighters are企equentlyencounteredbydangerofinhalingthetoxicgases， whichareproducedbysmo1deringafterextinguishingthefre.
M o s t c o m m o n l y i n f r r e s ， C O a n d H C N is a m a j o r t o x i c g a s Although C O has be en considered the primary cause of deathistoricaly，there is evidence 血at HCN is directly responsib1eformanymoredeathsthanwasprevious1yasumed The four gases，CO，HCN，nitric oxide (NO)and hydrogen
Corespondingauthor.Fax:+82-97-52・6075 E-mailadres:konishi@oita-c.tac.jp
su1fide (H2S) al readily inhibitoxygen consumption by mitochondria1cytochromeoxidase[2].
Toxicevaluationofregaseshavebencariedoutinmock-up bui1dingsand1aboratory scale combustion equipment using modelanirnalssuchasratsandmice[1].However，researches arenowatthefore企ontofaglobal仕endturningawayfrom usinganimalsforscientificandcomercialtests.Ensuringthat anirna1 experimentation must be caried out acording to 3R(Rep1acement，Refmement，Reduction)princip1es.Recently， Organization for Economic Co-operation and Deve10pment (OECD)testGuide1ineforTestingofChemica1s401[3]have revised into the new test guideline 420 [4]，in which they emphasized the reduce of the anima1s in experiments. Moreover，inJapan，universi句I orcolegeresearchcomite strictlyregulatesanirna1experirnentscausingpainandsu旺ering suchasthermalburnsandtoxicgasexposure.
The use ofhuman cels in vitro cytotoxicity 0百ers an alternativemethodto1aboratoryanima1s.A549，ahumanlung alveo1ar type I cel 1ine have a1ready ben eva1uated in numerousstudies，anddemonstratedtobeapractica1，re1iab1e andadvancedto1foridentificationof血ecytotoxice百ectsof chemical compounds[5四 7]. Recent1y， F. Lestari et a.l at Chemica1 Safety and Aplied Toxico1ogy Laboratories in Australiahavedeve10pedthealternativemethodforfresmoke to泊 city testing using A549 cel ls [8-10]. Their group has investigated the combustion to泊 city of po1ymers and compositesundernon-flarningcombustion
Since2002，our1aboratoryundergoesinterdiscip1inaryresearch onthermalengineringandmedicine[l1].Recentl)ら wehave startedthevitrocytotoxicityasesmentoffregasonA549 cels and developed fre gas exposure system.This paper reportstheexaminationofthetoxicityoftheeftluentsproduced 企omfourplastics:ABS，nylons，UrethaneandMelarnine
2 Experimentalapparatusandmethod
2.1 HumanA549lmigcel
A549cels企omahumanCaucasianlungadenocarcinomawith thealveolartypeIphenotypewereobtained企omDr.Kazuyo SenbaatBeppuUniversity.A549celsarehumanalveolarbasal epithelialcelsasshowninFig.l.Theyaresquarnousinnature and responsible for the di伍 lsion ofsubstances acros the alveoli of 1ungs. In vi仕o they grow adherently as a monolayer.The doubling time for the cel l was found to be aproximately 2 hrs.Fig.2 shows typica1 morphological diferencesbetwen(a)normalA549culturedwithCO2gas