GPO Box 58, Sydney NSW 2001, Australia

Existing Chemical HazardAssessment Report

DiisobutylPhthalateJune 2008

NATIONALINDUSTRIALCHEMICALSNOTIFICATIONANDASSESSMENTSCHEME

GPO Box 58, Sydney NSW 2001, Australia

©CommonwealthofAustralia2008

Thisworkiscopyright.Youmaydownload,display,printandreproducethismaterialinunalteredformonly(retainingthisnotice)foryourpersonal,non-commercialuseorusewithinyourorganisation.ApartfromanyuseaspermittedundertheCopyrightAct1968,allotherrightsarereserved.RequestsandinquiriesconcerningreproductionandrightsshouldbeaddressedtoCommonwealthCopyrightAdministration,AttorneyGeneral’sDepartment,RobertGarranOffices,NationalCircuit,BartonACT2600orpostedat

Preface

ThisreportwascompiledundertheNationalIndustrialChemicalsNotificationandAssessmentScheme(NICNAS).ThisSchemewasestablishedbytheIndustrialChemicals(NotificationandAssessment)Act1989(Cwlth)(theAct),whichcameintooperationon17July1990.

TheprincipalaimofNICNASistoaidin the protection ofpeopleat work,the public and theenvironmentfromtheharmfuleffectsofindustrialchemicals.

NICNASassessmentsarecarriedoutinconjunctionwiththeDepartmentofEnvironmentandHeritage,whichcarryouttheenvironmentalassessmentforNICNAS.NICNAShastwomajorprograms:theassessmentofthehealthandenvironmentaleffectsofnewindustrialchemicalspriortoimportationormanufacture;andtheotherfocussingontheassessmentofchemicalsalreadyinuseinAustraliainresponsetospecificconcernsabouttheirhealth/orenvironmentaleffects.

ThereisanestablishedmechanismwithinNICNASforprioritisingandassessingthemanythousandsofexistingchemicalsinuseinAustralia.

ForthepurposesofSection78(1)oftheAct,copiesofassessmentreportsforNewandExistingChemical assessmentsare freelyavailable fromthe web(CommonwealthChemicalGazette(

CopiesofthisreportandotherNICNASreportsareavailableontheNICNASwebsite.HardcopiesareavailablefromNICNASatthefollowingaddress:

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SydneyNSW2001AUSTRALIA

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Freecall:1800638528

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OtherinformationaboutNICNAS(alsoavailableonrequest)includes:

•NICNASAnnualReports.

•NICNASServiceCharter.

•BrochureonNICNASRegistration.

MoreinformationonNICNAScanbefoundattheNICNASwebsite:

Overview

Thisreviewofdiisobutylphthalate(DIBP)isahealthhazardassessmentonly.Forthisassessment,primaryreferenceswerethemainsourceofinformation.InformationwascollecteduptoSeptember2006.

AccordingtotheEuropeanCouncilofPlasticisersandIntermediates,DIBPisaspecialistplasticiseroftenusedincombinationwithotherhighmolecularweightphthalatesasagellingaid.IthasverysimilarapplicationpropertiestoDBPandmaythereforebeusedtosubstituteforDBPinmost,ifnotall,ofitsapplications.TheserangefromtheplasticisationofPVCtotheproductionofpaints,printinginksandadhesives.

InAustralia,DIBPisimportedforuseasaplasticiserforthemanufactureofPVCandrubber.Itisalsoimportedasacomponentofindustrialadhesivesandcatalystsystemsforpolypropyleneandfibreglassmanufacture.ImportedDIBPisalsosoldtovariousinstitutionsandlaboratoriesforresearchandproductdevelopment.

Structurally,phthalateestersarecharacterizedbyadiesterstructureconsistingofabenzenedicarboxylicacidheadgrouplinkedtotwoestersidechains.DiBPpossesses2branchedestersidechainseachwitha3carbonbackbone(C3).

ToxicitydataforDIBPwerenotavailableforallhealthendpoints.Forendpointswithmissingorincompletedata,informationfromstructurallysimilarphthalates,whereavailable,wasusedtoextrapolatepotentialtoxicity.Relevantread-acrossinformationwasobtainedfromotherNICNAShazardassessmentreportsforphthalatesandtheNICNASPhthalatesHazardCompendium,whichcontainsacomparativeanalysisoftoxicityendpointsacross24ortho-phthalates,includingDIBP.

DIBPappearstobereadilyabsorbedviatheoralanddermalroutes.Basedonadermalabsorptionstudy,DIBPundergoesprimarymetabolismintothehydrolyticmonoester,monoisobutylphthalate(MIBP),beforeexcretion.Urinewasthemajorrouteofexcretionwithminorbiliaryexcretionbeingobserved.Therewaslittleaccumulationintherattissues.

DIBPhasaloworderofacutetoxicitybytheoral,intraperitonealanddermalroute.DIBPisreportedtocauseminimalskinirritationinguineapigs.Noeyeirritationorskin sensitisationwasreportedinanimals.

A4-monthrepeateddosetoxicitystudyreportedlowbodyandtestesweightsandincreasedliverweightsinratswitha5%diet.TheNOAELwas1%indiet.

Basedonavailablestudies,thegenotoxicpotentialofDIBPcouldnotbedetermined.

NocarcinogenicitydataareavailableforDIBP.Duetoinsufficienttestingonotherphthalates,itwasnotpossibletoextrapolatethecarcinogenicpotentialforDIBP.

Withrespecttoreproductivetoxicity,aNOAELwasnotestablishedinanyoftheanimalstudies.AdministrationofDIBPathighdoses(approximately2000mg/kgbw/d),induceddecreasedbodyweightafter1weekoforaldosinginratsandmice.Relativetestesweightwasincreasedinmiceanddecreasedinratswhiletesticulartestosteronecontentwasdecreasedinbothspecies.TheLOAELfromthesestudieswas2000mg/kgbw/d,basedondecreasesinbodyweightandtesticularcontent.Inotherstudies,similarresultswereobtainedwhenratsandmicewerefeddietscontainingMIBP.

Limiteddevelopmentaltoxicitydatawereavailable.Inrats,oralexposuretoDIBPduringgestationwasassociatedwithcompletelossoflittersatmaterno-toxicdoses.Atlowerdoses,DIBPinduceddecreasedfoetalweightandincreasedincidenceofundescendedtestes.Inmalefoetusesatterm,DIBPdecreasedtesticulartestosteroneproductionexvivoandtestosteronelevelsintestesandplasma,decreasedanogenitaldistance(AGD),andinducedpathologicalchangesinthetestesincludingclusteringofsmallLeydigcellsandvacuolisationofSertolicells.TheNOAELwas250mg/kgbw/dbasedondecreasedpupweightandincreasedincidenceofundescendedtestes.

ArecenthumanstudyshowedurinaryMIBPconcentrationsinmotherswereinverselyrelatedtoanogenitalindex(AGI)inmaleoffspring.However,multipleexposurestodifferentphthalatesmayhavecontributedtothis effectandthereliabilityofAGIhasnot beenverifiedinhumans.

Table of Contents

PREFACEiii

OVERVIEWiv

ACRONYMSANDABBREVIATIONSvii

1.INTRODUCTION1

2.IDENTITY2

4.HUMAN HEALTH HAZARD4

4.1Toxicokinetics4

4.2Acute toxicity4

4.3Irritation5

4.3.1Skin irritation5

4.3.2Eye irritation5

4.4Sensitisation5

4.5Repeated dose toxicity6

4.6Genetic toxicity6

4.7Carcinogenicity7

4.8Reproductive toxicity7

5.HAZARD CHARACTERISATION11

6.HUMAN HEALTH HAZARD SUMMARYTABLE13

REFERENCES15

APPENDIX-ROBUSTSTUDY SUMMARIES17

AcronymsandAbbreviations

AGDanogenitaldistance

AGIanogenitalindex

bwbodyweight

CCelsius

CASChemicalAbstractsService

CHOChinesehampsterovary

dday

DBPdibutylphthalate

DEHPdiethylhexylphthalate

DIBPdiisobutylphthalate

DMSOdimethylsulfoxide

DNAdeoxyribonucleicacid

ERoestrogenreceptor

ffemale

F0parentalgeneration

F1filial1(firstgeneration)

F2filial2(secondgeneration)

ggram

GDgestationday

GLPgoodlaboratorypractice

hhour

ipintraperitoneal

kgkilogram

kPakilopascals

Llitre

LD50medianlethaldose

LOAELlowest-observed-adverse-effectlevelmmale

mgmilligram

MIBPmonoisobutylphthalate

mLmillilitre

NICNASNationalIndustrialChemicalsNotificationandAssessmentSchemeNOAEL no-observed-adverse-effectlevel

NTPNationalToxicologyProgram

ppmpartspermillion

PVCpolyvinylchloride

w/wweightperweight

μmicro

1.Introduction

Thisreviewofdiisobutylphthalate(DIBP)isahealthhazardassessmentonly.Forthisassessment,primaryreferenceswerethemainsourceofinformation.InformationwascollecteduptoSeptember2006.

Information onAustralianuseswascompiledfromdata suppliedbyindustryin 2004and2006.

Referencesnotmarkedwithanasteriskwereexaminedforthepurposesofthisassessment. References notexaminedbutquotedfrom thereferencesourcesassecondarycitationsarealsonotedinthisassessmentandmarkedwithanasterisk.

Hazardinformationfromthisassessmentispublishedalsointheformofaphthalatehazardcompendiumprovidingacomparativeanalysisofkeytoxicityendpointsfor24ortho-phthalateesters(NICNAS,2008).

2.Identity

2.1Identification of the substance

CASNumbers:84-69-5

ChemicalName:1,2-Benzenedicarboxylicacid,bis-(2-methoxypropyl)esterCommonName: Diisobutylphthalate(DIBP)

MolecularFormula:C16H22O4 StructuralFormula:

MolecularWeight:278.35

Synonyms:Phthalicacid,diisobutylester;Di(isobutyl)-1,2-benzenedicarboxylate

Purity/Impurities/Additives:Purity99%

2.2Physico-chemical properties

Table1:Summaryofphysico-chemicalproperties

s,viscousliquid

m3

Paat20°C

/L

Source:IPCS (2001), HSDB(2006)

-7atm.m3/moleosedcup)

3.Uses

AccordingtotheEuropeanCouncilofPlasticisersandIntermediates(ECPI,2006),DIBPisaspecialistplasticiseroftenusedincombinationwithotherhighmolecularweightphthalatesasagellingaid.IthasverysimilarapplicationpropertiestoDBPandmaythereforebeusedtosubstituteforDBPinmost,ifnotall,ofitsapplications.TheserangefromtheplasticisationofPVCtotheproductionofpaints,printinginksandadhesives.

InAustralia,DIBPisimportedforuseasaplasticiserforthemanufactureofPVCandrubber.Itisalsoimportedasacomponentofindustrialadhesivesandcatalystsystemsforpolypropyleneandfibreglassmanufacture.Imported DIBPisalso soldtovariousinstitutionsandlaboratoriesforresearchandproductdevelopment.

4.Human Health Hazard

4.1Toxicokinetics

Previous evaluations

Nodata

Data not reported in previous evaluations

ThedermalabsorptionofDIBPhasbeenassessed,alongwithotherphthalates(Elsisietal.,1989).FurfromanareaonthebackofmaleFischer344ratswasclippedand14Cphthalatediesterwasappliedatadoseof157µmol/kg.Urineandfaeceswerecollectedoverasevendayperiodandtheamountof14Cexcretedwastakenasanindexofthepercutaneousabsorption.Thecumulativepercentagedoseexcretedinsevendays forDIBPwasabout51% oftheapplied14C.UrinewasthemajorrouteofexcretionforDIBP,withsomeexcretioninthefaeces,presumablyduetobiliaryexcretion.Aftersevendays,thetotalrecoveryforDIBPwas93%.

Inhumans,DIBPismetabolisedtomonoisobutylphthalate(MIBP)whichcanbedetectedintheurine(Swanetal.,2005).Apartfromthisobservationinhumans,noinformationonmetabolismisavailable.

Conclusion

DIBPappearstobereadilyabsorbedviathedermalroute.Urinewasthemajorrouteofexcretionwithminorbiliaryexcretionbeingobserved.Therewaslittleaccumulation inthe rat tissues.In humans,DIBP undergoesprimarymetabolismintoMIBP,whichwasdetectedinurine.

4.2Acute toxicity

Previous evaluations

Table2:Summaryofacutetoxicitystudies

OralMouse12800 mg/kgbwEastmanKodak(1978)

Data not reported in previous evaluations

Nodata

Conclusion

DIBPhasaloworderofacutetoxicitybytheoralroute(LD50mouse=12800-39520mg/kgbw;LD50rat=16000-60320mg/kgbw),andintraperitoneal(i.p.)route(LD50mice=3990-12800mg/kgbw;LD50rat>1600mg/kgbw).

4.3Irritation

4.3.1Skin irritation

Previous evaluations

Nodata

Data not reported in previous evaluations

Lawrenceetal.(1975)reportednegativeresultsofirritationtestsonundilutedDIBPusingintradermalinjections.However,thetypeofanimalsusedwasnotstatedandlimitedinformationisprovided.

EastmanKodakCo.(1978)reportedthatDIBPwasaslightskinirritantinguineapigs.Nofurtherinformationisavailable.

Conclusion

DIBPhasbeenreportedtocauseminimalskinirritationinguineapigs,althoughnodataarepresented.

4.3.2Eye irritation

Previous evaluations

Nodata

Data not reported in previous evaluations

Lawrenceetal.(1975)reportednegativeresultsoftestsonundilutedDIBPintheeyesofrabbits.Nofurtherinformationisavailable.

Conclusion

DIBPwasreportednottocauseeyeirritationinrabbits.

4.4Sensitisation Previous evaluations

Nodata

Data not reported in previous evaluations

EastmanKodakCo.(1978)reportedthatDIBPwasnotaskinsensitiseringuineapigs.Nofurtherinformationisavailable.

Conclusion

DIBPhasbeenreportedtonotcauseskinsensitisationinguineapigs,althoughnodataarepresented.

4.5Repeated dose toxicity Previous evaluations

Nodata

Data not reported in previous evaluations

Hodge(1954)reportedonafourmonthrepeateddosedietarystudyinvolvingalbinorats(speciesnotprovided)(5/sex/group)fed0,0.1,1.0and5%DIBP(dosesinmg/kgbwnotprovided).Bodyweightsandhaematologicalparametersweremeasured.Organweightsweredeterminedatautopsy.Liversandkidneyswereexaminedhistologically.Significantdecreasedbodyweightswereobservedinbothsexesat5.0%(decreaseupto43%formalesand13%forfemalesat4months).Redbloodcellcountsinthe5%malegroupandhaemoglobinlevelsinbothsexesreceivingthisdosewereslightlyreduced.Theseeffectswerenotdose-related.Bothabsoluteandrelativetestesweightsinthe5%groupwereconsiderablyreduced.Nostatisticalanalyseswereconductedbutreductionswerenotedtoapproximately30%and50%ofcontrolvaluesrespectively.Absoluteandrelativeliverweightswereraised inthe 5% groups in bothsexes. Formales, absoluteweightswere increasedby5%;relativeweightsby80%.Forfemales,absoluteweightswereincreasedby40%;relativeweightsby60%.Pathologicalexaminationsofliverandkidneywereunremarkable.

Hodge(1954)alsoreportedonalimitedshorttermfeedingstudyindogs.Onemaleandonefemaledog(speciesunknown)werefedwithDIBPviadietatadailyrateof

0.1mL/kgfeedand2.0mL/kgfeedrespectivelyfor2months.Weightlosswasnoted

inthefemaledogbuthaematologicalandurineanalyseswereallnormal.Atstudytermination,therewasanincreaseinrelativeliverweightinthefemaledogcomparedtohistoricalcontrols.Nohistologicalchangesinliverwereobserved.Inthemale dog,histological examinationof testes revealedabnormallyfewsperm. Thestudywaspoorlyreported.Noconclusionscanbedrawnfromthisstudy.

Conclusion

The main targetorgan forDIBP following a4-month repeatdose toxicity study in ratwastheliver.TheNOAELwasdeterminedtobe1%(dietarylevel),withaLOAELof5%basedondecreasedbodyandtestesweightsandincreasedliverweights.

4.6Genetic toxicity

Previous evaluations

Nodata

Data not reported in previous evaluations

DIBPwasfoundnottobe mutagenicwithandwithoutS9activationinan8-azaguanineresistanceassayinSalmonellatyphimuriumTA100(concentrationnot

provided)(Seed,1982).ResultsofaSalmonellatyphimuriumassay(NTPTechnicalBulletin,1982)withS9activationwerenegative.Zeigeretal.(1985)tested34phthalatesandrelatedchemicals,includingDIBP,formutagenicityinSalmonellatyphimuriumstrainsTA98,TA100,TA1535andTA1537withandwithoutS9activation,upto10mg/plateunlesslimitedbysolubilityand/ortoxicity.DIBPwasnotmutagenic.

Kleinsasseretal.(2000a)reported,usinganinvitrocometassay,thatDNAdamage(single-strandbreaks)wassignificantlyinducedbyDIBP(354μmol/mL)inhumanoropharyngeal(n=40)andnasal(n=30) mucosasamples,ascomparedtothenegativecontrol(DMSO).

Infurtherwork,Kleinsasseretal.(2000b)foundthatDIBPinducedstrandbreaksinDNA,inbothbloodlymphocytesandnormalmucosalcellsfromtheoropharynxorlarynxof60humanpatientswithheadandneckcancer.

Conclusion

DIBPinducedDNAdamage(single-strandbreaks)inaninvitroCometAssay.Itwasnotmutagenicinbacterialmutationassays.

Noinvitrochromosomalaberrations,mammalianmutationandinvivogenotoxicitystudiesareavailable.Overall,thegenotoxicpotentialofDIBPcannotbedetermined.

4.7Carcinogenicity

Previous evaluations

Nodata

Data not reported in previous evaluations

Nodata

Conclusion

Nodata

4.8Reproductive toxicity

Traditionalhazardassessmentsconsidereffectsonfertilityseparatefromdevelopmentaltoxicity.Fertilityistestedbyexposingsexuallymatureadultstoachemicalandexaminingtheeffectsonreproductivecapacity.Developmentaltoxicityisstudiedbyexposingpregnantdamsandlookingforeffectsinthefoetuses.Chemicalsthataffectthedevelopingreproductivesystemfollowingprenatalexposuremayalsoaffectsexualmaturationorfunctionalreproductivedisordersthatareonlyapparent atmaturity. Developmentaltoxicity can thereforelead toeffects onfertilityandthetwoendpointscannotbeclearlydistinguished.

Inthishazardassessment,dataarepresentedonthebasisoftestprocedure.Testproceduresincluderepeatdosetoxicitystudiesthatdoseadultanimalsforvaryingdurations,prenataldevelopmentaltoxicitystudies(onlythedam isdosed,studyendsbeforeparturition) and postnatal developmental toxicitystudies (dam isdosed duringgestationandallowedtolitter,studyendsduringweaning).Theeffectsonfertility(asadults)anddevelopment(asfoetuses)arethendiscussedseparately.

Previous evaluations

Nodata

Data not reported in previous evaluations

Human studies

Associationbetween11maternalurinaryphthalatemonoesterconcentrationsandgenitalparameterssuchasanogenitalindex(AGI)[i.e.anogenitaldistance(AGD)normalised forbodyweight]andtesticulardescentinchildrenwasinvestigatedin85mother-sonpairs(Swanetal.,2005).UrinaryMIBPconcentrationwasinverselyrelatedtoAGI.ThisstudyhasbeencriticisedbyMcEwenetal.(2006)fromtheCosmeticandFragranceAssociationsofAmericaandEurope.TheysuggestedthatAGDismorelikelytobeproportionaltoheightratherthanweightandthatmaternalphthalateurinaryconcentrationswerenotnormalisedforurinevolume.Thereliabilityofthe measurementofAGDinhumanshasnotbeenverified.Onestudyof87neonatesthathasassessedthecorrelationofAGDwithbodyweightfoundinmales acorrelationof0.48 andthatbodylength maybea slightly betterpredictorforAGDthanweight(Salazar-Martinezetal.,2004).

Repeat dose toxicity studies

OishiHiraga(1980b,c)foundthatfeedingadietcontaining2%(approximately2000mg/kgbw/d)ofDIBPforaweekresultedinsignificantly(p<0.05)decreasedabsoluteandrelativeweightofthetestesinratsbutsignificantlyincreasedrelativetestesweight in mice(there was no difference in absolute testes weight). Zincconcentrationsinthetestesandliverweresignificantlydecreasedinbothspecies.Ongrossexamination,thetestesofDIBP-treatedratswerereducedinsizeandmicroscopicexaminationindicatedmarkedinhibitionofspermatogenesisanddesquamationofspermatocytes.

TheeffectsofMIBPonratandmousetesteshavebeenstudied(OishiHiraga1980a,d;Fosteretal.,1981).Micefeddietscontaining2%ofMIBPhadsignificantlyincreasedrelativetestesweightassociatedwithdecreasedbodyweight.Theaveragezincandtestosteronelevelsinthetestesoftreatedmiceweresignificantlylowerthanthecontrols.Inratsfeddietscontaining2%MIBP,bodyweightwas decreased aswas absoluteand relative testesweights. Testicularzincandtestosteroneconcentrationsaswellasserumtestosteroneconcentrationweresignificantlyreduced. Fosteret al.(1981) reportedthatwhenMIBPwasorallyadministeredtoyoungmaleratsat800mg/kgbw/dforsixdays,theanimalsdevelopedmarkedtesticularatrophyandzincmetabolismwasaltered,withincreasingurinaryexcretionofzincanddepletionofitsconcentrationintesticulartissues.

Prenatal developmental toxicity studies

Singhetal.(1972)administereddosesof0.375,0.75and1.25mL/kgbw(approximately390,780and1300mg/kgbw)ofDIBPbyintraperitoneal(ip)injectiontopregnantSpragueDawleyratsonGD5,10and15.Effectsobservedincludeddecreasedaverageweightoffoetusesatalldoselevelsandincreasednumbersofresorptionsandskeletalabnormalities(“partiallyelongatedandfusedribs”)atthehighestdose(1300mg/kgbw).Deadfoetuseswerefound.Thepossibleeffectonmaternalhealthwasnotdescribed.

FourgroupsofpregnantWistarratsweregavagedfromGD7toGD19or20/21witheithervehicle(cornoil)or600mg/kgbw/dofDIBP(Borchetal.,2006).AdministrationofDIBPresultedinsignificantreductioninAGDinmalepups(andincreasedAGDinfemalepups)atGD20/21togetherwithreductioninbodyweightsofmaleandfemalefoetusesandreductionsintesticulartestosteroneproductionandtesticulartestosteronecontentinthemaleoffspring.Testicularpathologicalchangeswerealsonoted:clusteringofsmallLeydigcellsonGD19orGD20/21andvacuolisationofSertolicellsonGD20/21.ANOAELwasnotestablished.

InastudyonSprague-Dawleyrats,DIBPwasadministeredtopregnantratbygavageatdosesof0(oliveoil),250,500,750and1000mg/kgbw/donGD6to20(Saillenfaitetal.,2006).Signsofmaternaltoxicitywereobserved,asevidencedbyreductioninbodyweightgain,atthebeginningoftreatment(GD6-9)at500mg/kgbw/dandhigherdosesalthoughoverallweightgaincorrectedforgraviduteruswasnodifferentthancontrolsattheendofgestation.Theincidencesofresorptionswassignificantlyincreasedat750mg/kgbw/d,andreached60%at1000mg/kgbw/d.Fortheoffspring,therewasadose-relateddecreaseinfoetalweight,whichwassignificantlylowerthancontrolfrom500mg/kg/d.Thereweresignificantincreasesintheincidenceoffoetuseswithskeletalmalformations(supernumerarylumbarandcervicalribs).Atthetwohighestdoses(750and1000mg/kgbw/d),theincidenceofmalefoetuseswithundescendedtesteswassignificantlyincreasedandthedegreeoftrans-abdominaltesticularmigrationwasincreasedinadose-relatedfashion,intreatedpups(significantfrom500mg/kgbw/d).TheNOAELformaternalanddevelopmentaltoxicitywas250mg/kgbw/d.TheLOAELwas500mg/kgbw/dbasedon theincreased incidence ofundescended testes and decreased weightin pupsanddecreasedbodyweightgaininadults.

Postnatal developmental toxicity studies

DIBP wasevaluatedinaChernoff-KavlockscreeningassayinwhichCD-1mice(50dams/group)weregavagedonGD6-13withasingledoselevelof4000mg/kgbw/dorcornoil(Hardinetal.,1987).Damswereallowedtolitterandapostnatalevaluationwasconducted.Atthatdose,nopregnantdamsgavebirthtoalivelitterand27exposeddamsdied.

Mode of action

DIBPwasnegativeforoestrogenicactivityinayeasttwo-hybridassay(Nishiharaetal.,2000)andshowedextremelyweakoestrogenicactivityinrecombinantyeastassay(Harrisetal.,1997).DIBP(upto10-5M)hadnobindingaffinityfortheoestrogenreceptorαorβinvitro(Todaetal.,2004)butwasalsofoundtoinduceoestrogenreceptor(ER)α-mediatedoestrogenicactivityandpossessantiandrogenicactivityinvitrobutshowednoactivitytowardsERβinCHO-K1cells(Takeuchietal.,2005).

Conclusion

Effectsonfertility

AdministrationofDIBPathighdose(approximately2000mg/kgbw/d)resultedindecreasedtestesweightinratsbutincreasedrelativetestesweightinmiceandinhibitionofspermatogenesisintherat.Thedevelopmentoftheseeffectswithina

weeksuggestsaspecificeffectratherthanasecondaryeffectofgeneralisedtoxicity(OishiHiraga,1980c).ANOAELwasnotestablished.

Effects on development

Arecenthumanstudy(Swanetal.,2005)showedurinaryMIBPconcentrationwasinverselyrelatedtoAGI.However,multipleexposurestodifferentphthalatesmayhavecontributedtothiseffect. Inaddition,theendpointmeasured, AGI,hasnotbeenverifiedinhumans.

Limiteddevelopmentaltoxicitystudiesareavailable.Inrats,oralexposuretoDIBPduringgestationwasassociatedwithcompletelossoflittersatmaterno-toxicdoses.ANOAELof250mg/kgbw/dandaLOAELof500mg/kgbw/dwereestablished,basedondecreasedpupweightandincreasedtransabdominalmigrationoftestes(Saillenfaitetal.,2006).Inanotherexperiment,malefoetusesattermdemonstrateddecreasedtesticulartestosteroneproductionexvivo,decreasedtestosteronelevelsintestesandplasma,decreasedAGD,andpathologicalchangesinthetestesincludingclusteringofsmallLeydigcellsandvacuolisationofSertolicellsat600mg/kgbw/d(Borchetal.,2005,2006).

Table4:Summaryofreproductivetoxicitystudies

NE=notestablished;↓=decreased;↑=increased;wt=weight

5.HazardCharacterisation

ToxicitydataforDIBPwerenotavailableforallhealthendpoints.Forendpointswithmissingorincompletedata,informationfromstructurallysimilarphthalates,whereavailable,wasusedtoextrapolatepotentialtoxicity.Relevantread-acrossinformationwasobtainedfromotherNICNASassessmentreportsforrelevantphthalatesandtheNICNASPhthalatesHazardCompendium(NICNAS,2008),whichcontainsacomparativeanalysisoftoxicityendpointsacross24ortho-phthalateesters,includingDIBP.DIBPhasalinearportionofcarbonsidechainof3carbonatomsinlength(branchedatC2).

DIBPappearstobereadilyabsorbedviathedermalroute.Itundergoesprimarymetabolismintothehydrolyticmonoester,MIBP,beforeexcretion.Urinewasthemajorrouteofexcretionwithminorbiliaryexcretionbeingobserved.Therewaslittleaccumulationintherattissues.

DIBPhasaloworderofacutetoxicitybytheoral,intraperitonealanddermalroute.DIBPisreportedtocauseminimalskinirritationinguineapigs.Noeyeirritationorskinsensitisationwasreportedinanimals,however,nofurtherinformationwasavailable.

A4-monthrepeateddosetoxicitystudyreportedlowbodyandtestesweightsandincreasedliverweightsinratswitha5%diet.TheNOAELwas1%indiet.Nootherdetailsregardingdoselevelswereavailable.DIBPwasnotmutagenicinbacterialmutationassaysbutthereisevidencethatitinducedDNAdamageinhumancellsinvitro.Noinvitrochromosomalaberrations,mammalianmutationandinvivogenotoxicitystudiesareavailable.Overall,thegenotoxicpotentialofDIBPcannotbedetermined.

NocarcinogenicitydataareavailableforDIBP.Duetoinsufficienttestingonotherphthalates,itisnotpossibletoextrapolatecarcinogenicpotentialforDIBP.

Withrespecttoreproductivetoxicity,DIBPinduceddecreasedbodyweightafter1weekoraldosinginratsandmiceaswellaseffectsontestesweightandtestosteronecontent.Relativetestesweightwasincreasedinmiceanddecreasedinratswhiletesticulartestosteronecontentwasdecreasedinbothspecies.SimilarresultswereobtainedwhenratsandmicewerefeddietscontainingMIBP.ANOAELwasnotestablishedinanyoftheanimalstudies.

Limiteddevelopmentaltoxicitystudiesareavailable.Inrats,oralexposuretoDIBPduringgestationwasassociatedwithcompletelossoflittersatmaterno-toxicdoses.Atlowerdoses,DIBPinduceddecreasedfoetalweightandincreasedincidenceofundescendedtestes.InmalefoetusesattermDIBPdecreasedtesticulartestosteroneproductionexvivoandtestosteronelevelsintestesandplasma,decreasedAGD,andinducedpathologicalchangesinthetestesincludingclusteringofsmallLeydigcellsandvacuolisationofSertolicells.TheNOAELwas250mg/kgbw/dbasedondecreasedpupweightandincreasedincidenceofundescendedtestes.

ArecenthumanstudyshowedurinaryMIBPconcentrationsinmotherswereinverselyrelatedtoAGIinmaleoffspring.However,multipleexposurestodifferent

phthalatesmayhavecontributedtothiseffect.Inaddition,thereliabilityofendpointmeasured,AGI,hasnotbeenverifiedinhumans.

AlthoughdataforDIBParelimited,thefertilityanddevelopmentaleffectsobservedaresimilartothosephthalateswithsidechainbackboneofcarbonsidechainsof4-6carbonatomsinlength(C4-6)(NICNAS,2008).TheseC4-6phthalatespreviouslyreferredtoas‘transitional’phthalates(PhthalateEstersPanelHPVTestingGroup,2001)havealsobeenassociatedwithmalereproductive(decreasedintesticulartestosteroneproduction)anddevelopmental(decreasedAGDandpathologicalchangesinthetestes)effects.Therefore,itcouldbearguedthatDIBPhasasimilarreproductivetoxicityprofileto‘transitional’(C4-6)phthalatesforwhichreproductiveanddevelopmentaleffectsarerecognised.

6.Human Health Hazard Summary Table

↓:decrease;↑:increase

Pageintentionallyblankfordouble-sidedprinting.

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Appendix- Robust Study Summaries

A1 - Genetic toxicity/carcinogenicity

Testsubstance : Diisobutylphthalate(DIBP).

Species:Specimensfromhumanmucosaoftheupperaerodigestivetract,harvestedduringsurgery.

Routeofadmin. : CellaliquotsincubatedwithDIBP.

Exposureperiod : Incubationtime60minutes.

StudyDuration:N/AFrequencyoftreatm.:N/APostexposureperiod : N/A

Doses : 130,216,354,650µmol/mL.

Controlgroup : Positiveandnegativecontrolchemicalswereused.

Guidelines : None

NOAEL/NOEL : N/A

LOAEL/LOEL : N/A

Method : The microgel electrophoresis technique (comet assay)

wasappliedtodetectDNA-strandbreaksinsinglecells.TheextentofDNAdamagewasquantifiedusingthe“Olivetailmoment”(OTM),whichisdefinedasthepercentageofDNAinthetailofthecometmultipliedbythemigrationdistance.

Results : Inn=40samplesoforopharyngealmucosa,DIBPat354

µmol/mLproducedanaverageOTMof9.6±5.8(negativecontrols,1.3±0.4;positivecontrols,63.5±

11.1).

Inn=30samplesofbiopsiesfromtheinferiornasalturbinates,DIBPat354µmol/mLproducedanaverageOTMof13.4±5.7(negativecontrols,1.6±1.1;positive

controls,55.7±10.6).

Conclusion : DIBP caused DNA strand breaks in isolated human

mucosalcellsfromtheupperaerodigestivetract.

Reference : Kleinsasser NH, Kastenbauer ER, Weissacher H,

MuenzenriederRKHarreusUA(2000a).Phthalatesdemonstrategenotoxicityonhumanmucosaoftheupperaerodigestivetract.EnvironmentalandMolecularMutagenesis35,9-12.

A2 - Reproductive toxicity

Testsubstance:Diisobutylphthalate(DIBP)

Type:Dietary administration of test material to assesseffectsonreproductiveorgans.

Species:JCL:Wistaryoungmaleratswereused,weighing

90-120g(mean108g,5weeksold).

Routeofadministration

:Dietary

Exposureperiod:Oneweek

StudyDuration:N/A

Frequencyoftreatment

Prematingexposureperiod

:Adlibitum

:N/A

Durationoftest:N/A

Doses:TwopercentDIBPinthediet(approx.2000mg/kgbw/d)

Controlgroup:Agroupfedthebasaldiet.

NOAELparental:N/A

NOAELF1offspring :N/A

Other:systemiceffects

:N/A

Guidelines:None

GLP:None

Method:Ten rats were given diets containing 2% DIBP.

Twentyratswereinthecontrolgroup.Bodyweightandfoodconsumptionweremeasureddaily.After

oneweekoftreatment,theratswerekilledbydecapitation,samplesofbloodwerecollected,andthefreshweightsofthetestes,liver andkidneyswereobtained.Serumandtissuesampleswerestored

at-80°Cuntilanalysis.

Result: Attheendof theexperiment,themeanbodyweightsofDIBP-treatedratswereslightlylowerthanthatofcontrols,butnotsignificantlydepressed.Foodconsumptionwasdecreasedduringthefirstthreedaysandthenrecoveredtothecontrol level.Absoluteandrelativeliverweightswereincreasedintreatedrats.Absoluteandrelativetesticularweightsweresignificantlydecreasedandthetestesweredecreasedinsize,withhistologicalexaminationrevealing adecreaseinspermatogoniaandspermatocytes.Therewasasignificantincreaseintesticular testosterone in treated rats but no

differenceinserumdihydrotestosteronelevels.Zincconcentrationsinthetestesandliverweredecreased.

Conclusion : TheeffectsofDIBPonthetestes(decreaseinorgan

weight,sizeandhistologicalchanges)inthisstudyinvolvedhighdosesbuttheirdevelopmentwithinaweeksuggestsaspecificeffectratherthanasecondaryeffectofgeneralisedtoxicity.

Reference : Oishi S Hiraga K (1980c). Testicular atrophy

inducedbyphthalateacidesters:Effectontestosteroneandzincconcentrations.Toxicologyand

AppliedPharmacology53,35-41.

A3 - Developmental toxicity/teratogenicity

Testsubstance : Diisobutyl phthalate

Species:Adult,virginfemaleratsoftheSprague-Dawleystrain,weighingbetween200and250g.Maleratsofthesamestrainandagewereusedformating

Routeofadmin. : Intraperitonealinjections.

Exposureperiod : Intraperitonealinjectionsweregivenondays5,10and15ofgestation

StudyDuration : 2oestruscyclespriorto mating–GD20

Frequencyoftreatm. : Asabove

Doses : Approx.390,780and1300 mg/kg bw.

Controlgroup : Cottonseedoil,normalsaline,distilledwaterand

untreatedcontrolsemployed

NOAELmaternaltox.:NotdeterminedNOAELteratogen. :NotdeterminedGuidelines : Nil

GLP : Nil

Method:Alltreatmentswereadministeredbyintraperitonealinjectionsondays5,10and15ofgestation.Onday20,eachratwaskilledwithether.Theuterinehornsandovariesweresurgicallyexposedtopermitcountingandrecordingofthenumbersofcorporalutea,resorptionsites,andviableanddeadfoetuses.Foetuseswereweighedandexaminedforgrossmalformations.Aproportionoffoetuseswerepreparedtopermitvisualisationof theskeletalsystem.

Results:Effectsobservedwerestatisticallysignificantdecreasesintheaverageweightoffoetusesatalldoselevelsandincreasednumbersofresorptionsandskeletalabnormalities(“partiallyelongatedandfusedribs”)atthehighestdose.

Conclusion:Decreasedaveragefoetalweightisindicativeof

developmentaltoxicity.

Reliability:Thestudyisoldandnotofastandarddesignanddoseswereadministeredintraperitoneally.Theeffectobserved(decreasedfoetalweight)werecomparedtountreatedcontrolsratherthanvehiclecontrol.

ReferenceSingh AR, Lawrence WH Autian J (1972).

Teratogenicityofphthalateestersinrats.JournalofPharmaceuticalSciences61(1),51-5.

Testsubstance:Diisobutylphthalate

Species:Sprague-Dawleyratsapproximately180-200g.

Routeofadmin.:gavage

Exposureperiod:GD6toGD20

StudyDuration:GD0toGD21

Frequencyoftreatm.:Asabove

Doses:250,500,750and1000mg/kgbw/d

Controlgroup:OliveoilNOAELmaternaltox.:250mg/kgbw/dNOAELteratogen. : 250mg/kgbw/dGuidelines : Nil

GLP:Nil

Method:PregnantfemalesweregivendailydosesofDIBPbygastricintubationfromGD6toGD20.Allfemaleswereobserveddailyforclinicalsignsoftoxicitywith bodyweightrecorded. OnGD 21,thefemalesweresacrificed.Uterinecontentswereexaminedtodeterminethenumberofimplantationsites,resorptions,anddeadandlivefoetuses.Alllivefoetuseswereweighted,andexaminedforexternalanomaliesincludingthoseoftheoralcavity.Halfwereexaminedforinternalsofttissuechanges andtheotherhalfforskeletalexamination.

Results:Signsofmaternaltoxicitywereobserved,asevidencedbyreductioninbodyweightgain,atthebeginningoftreatment(GD6-9)at500mg/kgbwandhigherdoses.Theincidencesofresorptionswassignificantlyincreasedat750mg/kgbw,andreached60%at1000mg/kgbw.Fortheoffspring,therewasadose-relateddecreaseinfoetalweight,

whichwassignificantlylowerthancontrolfrom500mg/kg/d.Thereweresignificantincreasesintheincidenceoffoetuseswithskeletalmalformations(supernumerarylumbarandcervicalribs).Atthetwohighestdoses(750and1000mg/kgbw/d),theincidenceofmalefoetuseswithundescendedtesteswassignificantlyincreasedandthedegreeoftrans-abdominaltesticularmigrationwasincreasedinadose-relatedfashion,intreatedpups(significantfrom500mg/kgbw/d).

Conclusion:Decreased average foetal weight and increased

incidenceofundescendedtestesareindicativeofdevelopmentaltoxicity.

Reference Saillenfait Am, Sabaté JP, Gallissot F (2006)Developmentaltoxiceffectsofdiisobutylphthalate,the methyl-branchedanalogueofdi-n-butylphthalate.ToxicologyLetters,165,39-46.