Existing Chemical HazardAssessment Report
DiisobutylPhthalateJune 2008
NATIONALINDUSTRIALCHEMICALSNOTIFICATIONANDASSESSMENTSCHEME
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©CommonwealthofAustralia2008
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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(
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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
2.1 / Identification of the substance / 22.2 / Physico-chemical properties / 2
3. / USES / 3
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
(1975)Intraperitoneal / Mouse / 6400-12800mg/kgbw / EastmanKodak(1978)
Intraperitoneal / Rat / >1600mg/kgbw / EastmanKodak(1978)
Oral / Rat / 60320mg/kgbw / Hodge(1954)
Oral / Mouse / 39520mg/kgbw / Hodge(1954)
Oral / Rat / 16000-28000mg/kgbw / EastmanKodak(1978)
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
Studytype / Route / Doses(mg/kgbw/d) / NOAEL
(mg/kgbw/d) / LOAEL(mg/kgbw/d)endpoint / Reference
Rats / ip / 0.375, 0.750,1.250 / NE / 375;Foetotoxicity / Singh etal.,
GD 5-15 / mL/kgbw (0,390, / 1972
780, 1300)
Rats,Wistar, / diet / 0,2% (0, 2000) / NE / 2000;↓testeswt,↓ / Oishi
male / testicular / Hiraga,1980c
10/group
7days / testosterone,↓liver
& testes zinc,
spermatogenesis and
desquamationof
spermatocytes
Mice / gavage / 0, 4000 / NE / 4000; noviable litters / Hardin et al.,
50/group / (1987)
GD 6-13
Mice, JCL: ICR, / diet / 0,2% (0,4000) / NE / 4000;↓bodywt / Oishi
male
10/group 7days / gain,↑testeswt,no effecton testosterone / Hiraga,
(1980b*)
Rats, Sprague / gavage / 0, 250,500, 750, / 250 / 500;↓foetalwt,↑ / Saillenfaitet
Dawley / 1000 / incidenceof / al., 2006
24/group / undescended testes
GD 6-20
Wistarrats / gavage / 0, 600 / NE / 600;↓testicular / Borch et al.,
10-12/group / testosterone content; / 2006
GD 7-19/21 / ↓testicular
testosterone
production;↓AGD
(absoluterelative,
male),↓bodyweight
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
Phthalate / Acute / IrritationSensitisation / RepeatedDoseToxicity / GeneticToxicity / Carcino-genicity / Fertility / DevelopmentalToxicityDiisobutyl / Oral / Skin irritation: / Rat: / In vitro: / Nodata / Male reproduction study / Developmentalstudy
phthalate / Rat: / minimal effects / NOAEL=1%(mg / Negative in / Rat: / Rat:
(DIBP) / LD50 =16000- / equivalents not / bacterial / NOAEL= notestablished / NOAEL
60320mg/kgbw / Eye irritation: / available) / mutation / Devp= 250 mg/kgbw/d
negative / assays / LOAEL= 2000 mg/kg
Mouse: / LOAEL =5%(mg / bw/d / LOAEL
LD50 =12800- / Skin / equivalents not / Positive in / ↓testesweightand / Maternal =500 mg/kg
39520mg/kgbw / Sensitisation: / available): / comet (DNA / histological changes / Devp= 500 mg/kg
negative / ↓bodyandtestes / damage) assay / ↓pupweightand↑trans-
Dermal / weights(m);↑ / using human / abdominal testes migration
Nodata / liverweights (m-f) / mucosa
(sample size =
Inhalation / 70)
Nodata
Invivo:
Nodata
↓: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.