Sheil et al. - 2021 - Topical Application of Lidocaine and Bupivacaine t

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ArticleTopicalApplicationofLidocaineandBupivacainetoDisbuddingWoundsinDairyCalves:Safety,ToxicologyandWoundHealingMeredithSheil1,* ,MichaelChambers2,AdamPolkinghorne3,4andBrendanSharpe2 Citation:Sheil,M.;Chambers,M.;Polkinghorne,A.;Sharpe,B.TopicalApplicationofLidocaineandBupivacainetoDisbuddingWoundsinDairyCalves:Safety,ToxicologyandWoundHealing.Animals2021,11,869.https://doi.org/10.3390/ani11030869AcademicEditor:AlisonSmallReceived:22January2021Accepted:16March2021Published:18March2021Publisher’sNote:MDPIstaysneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffil-iations.

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1Accepted:16March2021Published:18March2021Publisher’sNote:MDPIstaysneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffil-iations. Copyright:©2021bytheauthors.LicenseeMDPI,Basel,Switzerland.ThisarticleisanopenaccessarticledistributedunderthetermsandconditionsoftheCreativeCommonsAttribution(CCBY)license(https://creativecommons.org/licenses/by/4.0/).1AnimalEthicsPty.Ltd.,YarraGlen3775,Australia2InvetusPty.Ltd.,Armidale2350,Australia;mchambers@invetus.com(M.C.);bsharpe@invetus.com(B.S.)3DepartmentofMicrobiologyandInfectiousDiseases,NSWHealthPathology,NepeanBlueMountainsPathologyService,Penrith2751,Australia;adam.polkinghorne@health.nsw.gov.au4NepeanClinicalSchool,FacultyofMedicineandHealth,UniversityofSydney,Kingswood2747,Australia*Correspondence:mlksheil@me.comSimpleSummary:Disbuddingisacommon,butpainfulprocedureperformedoncalvestopreventhorngrowth.Tri-Solfen®isacombinationlocalanaestheticandantisepticformulationwhich,appliedtopicallytothedisbuddingwound,isreportedtoreducecalfpain.Appliedinthismanner,thelocalanaestheticsinTri-Solfen®,lidocaineandbupivacaine,arereportedtobepoorlyabsorbed,resultinginlowriskofneurologicalorcardiotoxiceffects.Thepotentialimpactsonotherblood,urineandtissueparametersandonwoundhealingwhenusedinthismanner,and/oraccidentaloverdosesituationsareunknown,however.Weperformedexperimentsinvestigating(i)thesafetyofTri-Solfen®(includingoverdosesituations)and(ii)theimpactofTri-Solfen®ondisbuddingwoundhealingunderfieldconditions.NoadversehealtheffectswereobservedinTri-Solfen®-treatedanimals,eventhosereceiving5 therecommendeddose,withnoclinicallysignificantdifferencesinmeasuredparametersbetweenplaceboandTri-Solfen®groups.Nonegativeimpactsonwoundhealingwerenoted.Conversely,lowerlevelsofbacterialwoundcolonisationwereevident,andtherewasreducedincidenceofabnormalwoundsatdays11–12inTri-Solfen®-treatedanimals.Abstract:Tri-Solfen®isacombinationtopicalanaestheticandantisepticsolutioncontaininglidocaine,bupivacaine,adrenalineandcetrimide.Appliedtowounds,itisreportedtoreducethepainexperi-encedbycalvesfollowingthermocauterydisbudding.Whilelidocaineandbupivacainearewidelyusedinmedicine,conflictingdataexistontheimpactofthesecompoundswhenapplieddirectlytothesurgicalwound.ToinvestigatethesafetyofTri-Solfen®appliedtothermocauterydisbuddingwoundsofcalves,experimentswereperformedtomeasure(i)thesafetyofTri-Solfen®(includinginoverdosesituations);and(ii)theimpactofTri-Solfen®applicationatrecommendeddosesondisbuddingwoundhealingunderfieldconditions.Haematological,biochemicalandurinalysisparametersdidnotshowclinicallysignificantdifferencesbetweenplaceboandTri-Solfen®groups(1 ,3 and5 dose).Noadversehealthimpactswerereported.HistopathologicalanalysisofwoundsnotedareductioninbacterialcoloniesinTri-Solfen®-treatedwounds.Underfieldconditions,nonegativeimpactsonwoundhealingwerenoted.Conversely,therewasreducedincidenceofabnormalwounds,withanassociatedtrendtowardimprovedaveragedailygainatdays11–12inTri-Solfen®-treatedanimals.Thesedataareconsideredtosupportthesafetyoftopicalanaesthesia,asformulatedinTri-Solfen®,tothethermocauterydisbuddingwoundincalves.Keywords:localanaesthetic;animalhusbandry;thermocautery;woundinfections;antiseptic

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asformulatedinTri-Solfen®,tothethermocauterydisbuddingwoundincalves.Keywords:localanaesthetic;animalhusbandry;thermocautery;woundinfections;antiseptic 1.IntroductionDisbuddingisananimalhusbandryprocedureconductedon-farmaspartofrou-tinemanagementincattleproductionenterprises[1].Removalofthehornbudand Animals2021,11,869.https://doi.org/10.3390/ani11030869https://www.mdpi.com/journal/animals

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Animals2021,11,869 2of17

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Animals2021,11,869 2of17 horn-buddingcellsisconsideredtocausesignificantpain,basedonavarietyofout-comemeasures[2–6].Theuseoftopicalanaesthesia,appliedtothedisbuddingwoundimmediatelypost-procedure,isemergingasasimpleandpracticalmethodtoaddresspost-operativepain.Tri-Solfen®,acombinationtopicalanaestheticandantisepticformu-lation,isalicensedproductinAustraliaandNewZealand,withademonstratedabilitytoreducepost-operativehyperalgesicresponsesandpain-relatedbehaviourincalvespost-disbudding[7,8]andcastration[9].Itcontainslocalanaesthetics(50g/LLidocainehy-drochlorideand5g/LBupivacainehydrochloride),avasoconstrictor(0.048g/LAdrenalineacidtartrate)andanantisepticagent(5g/LCetrimide).Theveterinarymedicinesregulatoryapprovalprocessrequires,amongstotherthings,highstandardsofproofofsafetyofnewanimalmedicines.Typically,thisinvolves“Tar-getAnimalSafety”,aswellas“field”safetystudies,forwhichthereareinternationallyharmonized(VICH)approvedstandardsandguidelines[10].Theformerisdesignedtoexaminedetailedbiochemical,haematologicalandhistopathologicalimpactsandalsoexaminetheimpactofmuchhigherdosages(e.g.,3–5 dosage)tounderstandtheriskofpotentialmisuseoraccidentaloverdose.Thelatterarelargerscaletrialsdesignedtoexaminesafetyofuseinthefield.Thedatageneratedfromsuchtrialscanfillinformationgapsandbeofhighpublicvalue.ThisisparticularlythecaseintermsofTri-Solfen®applicationtoopenwounds,suchasthedisbuddingwoundincalves.AlthoughthelocalanaestheticsinTri-Solfen®havebeenwidelyusedinhumanandveterinarymedicineformanydecades,thereremainsadearthofinformationofthesafetyimpacts,bothlocalandsystemic,whenusedincalves,andwhenappliedtopicallytosignificantopenwounds.Theacutesystemictoxiceffectsoflidocaineandbupivacainehavebeenwelldescribedinhumansandavarietyofspecies(e.g.,dogs,goatkids,lambs,pigs,horsesandmon-keys)[11].Lidocaineandbupivacaineareamidelocalanaestheticsthatalterneuralsignalconductionbyblockingthefastvoltage-gatedNa+channelsintheneuronalcellmembraneresponsibleforactionpotentialpropagation.Acutesystemictoxiceffectsmostcommonlyoccurinthesettingofrapidintravascularabsorptionandprimarilymanifestinthecentralnervoussystem(CNS)and/orcardiovascularsystem(CVS)athigherdoses.Althoughtheriskofacutesystemictoxicityfollowingtopicalapplicationtointactskinislowduetopoorpenetrationandlowabsorption[12],greaterabsorptionandtoxicityriskmayoccuriftheagentisappliedtomucusmembranesoropenwounds[13,14].Inviewofthisrisk,untilrecently,topicallocalanaestheticshavebeeninfrequentlyemployedonsignificantopenwounds,resultinginalackofavailablesafetydatafollowinguseinthissetting.Furthermore,inviewofthefactthatbothlidocaineandbupivacainewereintroducedwellbeforetoday’sstandardsofsafetytesting,thereisapaucityofavailabledataonpotentialimpactsonhaematologicalandbiochemicalparametersand/orotherorgansystems.Tobe-gintoaddressthesedatagaps,werecentlyinvestigatedthepharmacokineticsoftopicaladministrationoflidocaineandbupivacainewithadrenaline(ascontainedinTri-Solfen®),tocalfdisbuddingwoundsimmediatelyfollowingthermocautery[15].Thisstudyfoundthattheselocalanaestheticsarepoorlyabsorbedthroughthedisbuddingwoundwithpeakplasmalevelsremainingwellbelowtoxicthresholdsanddecliningquicklyby48hpost-administration.NoclinicalsignsofCNSorCVSinvolvementwereobservedinanyanimalsthroughouttheinvestigation.Theseresultsindicateverylowriskoflocalanaes-theticsystemictoxicityfollowingtopicalapplicationofTri-Solfen®tocalvesfollowingthermocauterydisbuddingattherecommendeddosagelevels.Dataonwiderpotentialtoxiceffectsincalves,orpotentialtoxicityathigherlevelsofdosingremainlacking.Questionsalsoremainoverpotentiallocaltoxicimpactsattheapplicationsitewhentopicallocalanaestheticsareappliedtoopenwounds.Bothanaestheticsmayinducecytotoxiceffectsifdeliveredinhighconcentrationstosensitivetissues(e.g.,corneaandcartilage[16])whichmayhaveramificationsfortheirapplicationtowoundsandthepotentialimpactsonwoundhealing.Intermsofwounds,invitrostudieshaveshownthatbothlocalanaestheticspreventcellgrowthandcausecelldeathatdilutionsusedincommerciallyavailablepreparations[17,18].Invivodataaremoreconflictinghowever,

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Animals2021,11,869 3of17

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Animals2021,11,869 3of17 withsomestudiesshowingtreatmentwithlocalanaestheticsmaysloworpreventheal-ing[19–22],whileothersreportnoeffect[22–26],or,indeed,showabeneficialeffect[27–29].Itispertinenttonotethatdetrimentaleffectsonwoundhealinggenerallyhaveonlybeenreportedfollowinginjectedratherthantopicalanaestheticapplication.Injectionsmaydisrupttissueplanesanddeliverlocalanaestheticsintotissuesunderpressure,resultinginmechanicaltraumathatdoesnotoccurwithtopicalapplication.Furthermore,invivo,anydeleteriouseffectsoflocalanaestheticsappliedtowoundssecondarytocytotoxicityhavethepotentialtobecounterbalancedbybeneficialeffectssuchastheirpotentialanti-inflammatoryand/orantimicrobialproperties.Bothbupivacaineandlidocainearereportedtohaveantimicrobialandinflammatoryactivityattherapeuticdoses[30].Accumulatingdatasuggestthatlocalanaestheticsmaypossessawiderangeofanti-inflammatoryactionsthroughtheir“stabilising”effectsoncellsoftheimmunesystem,aswellasonothercells(e.g.,microorganisms,thrombocytesanderythrocytes)[31].Althoughthedetailedmechanismsofactionarenotfullyunderstood,theyhaveprovedtobeverysuccessfulinthetreatmentofburninjuries[31–34],andarereportedtoreduceriskofthrombo-embolismpost-surgery[35].Interestingly,invitrostudieshaverevealedthatlocalanaestheticsincommerciallyavailablesolutionsalsohavebacteriostaticand/orbacte-ricidalactivityagainstequinebacterialpathogens[36].Invivostudies,however,castsomedoubtontheclinicaleffectivenessoflidocaineandbupivacaineasantimicrobialagents.NosignificantdifferenceswereobservedintheinflammatoryresponseorviablebacterialcountsofrabbitsexperimentallyinoculatedwitheitherEscherichiacoliorStaphylococcusaureusandtreatedwitheither0.9%NaCl,bupivacaineorlidocaine[37].Tri-Solfen®containsanantisepticaswellasthelocalanaestheticactives.TheimpactofTri-Solfen®onwoundhealinghasbeenreportedinarangeofwoundtypesindifferentspecies.Lomaxetal.reportedanimprovedrateofhealingoverthefirst14daysinlambsfollowingtaildockingandtheflystrikepreventionprocedureofmulesing[38].Sutherlandetal.[39]reportedthatpigletcastrationwoundstreatedwithTri-Solfen®healedaswellasthosetreatedwithantisepticalone,andbetterthanthosetreatedwithadifferentlocalanaesthetic,withoutantiseptic.Mostrecently,Tri-Solfen®treatmentofdisbuddingwoundsincalvessuggestedthatitsusemayenhancewoundhealingcomparedtothealternativeantimicrobialspray[40].ItisestimatedthatTri-Solfen®hasnowbeenusedon>100millionanimals,withoutreportsofanegativeimpactonwoundhealing,includinginarangeofstudiesreportingitsuseinsheep[28,38],cattle[9]andpigs[41,42];however,histopathologicalfindingshavenotbeenreported.Inthecurrentstudy,wereporttheresultsofstudiesexamining(a)thetargetanimalsafetyand(b)fieldsafetyoftheapplicationofTri-Solfen®tocalfdisbuddingwoundsincludingitsimpactsonwoundhealingand/orbacterialcolonisationandinfections.2.MaterialsandMethods2.1.StudyDesignAssessmentofthesafety,toxicityandwoundhealingimpactsofTri-Solfen®(50g/LLignocainehydrochlorideand5g/LBupivacainehydrochloride,0.048g/LAdrenalineacidtartrate,5g/LCetrimide)administeredtodisbuddingwoundsincalveswereevaluatedintwomajorexperiments.Study1wasablinded,randomised,controlled,parallelgroup“TargetAnimalSafety”(VICH-GL43)study.ThestudywasdesignedtoinvestigatethesafetyofTri-Solfen®appliedtocalfdisbuddingwounds,includingatdosesupto5 therecommendeddose.ThestudywasdesignedinaccordancewithGoodLaboratoryPractice(GLP)[43–46]andtargetanimalsafetystudy[10]guidancedocuments.EthicsapprovalwasprovidedbytheUniversityofNewEnglandAnimalEthicsCommittee,Armidale,Australia(19-078).Study2wasablinded,randomised,controlledgroupfieldsafetyandefficacytrial.Thisstudywasdesignedtoinvestigatethesafety,includingwoundhealingimpacts,ofTri-Solfen®appliedattheupperrecommendeddosetocalfdisbuddingwoundsunderfieldconditions.Thestudycompliedwiththefollowingnationalandinternationalstandards:

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Animals2021,11,869 4of17

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Animals2021,11,869 4of17 (i)VICHGL9—GoodClinicalPractice;(ii)APVMADataGuidelines—Efficacyandtargetanimalsafetygeneralguidelines.ApprovalforthisprojectwasprovidedbytheUniversityofNewEnglandAnimalEthicsCommittee,Armidale,Australia(19-095).Withregardtoanimalwelfare,itisnotedthatcalfdisbuddingispracticedunderarangeofdifferentanalgesicoptionsindifferentjurisdictionsglobally.Althoughrec-ommended,theuseofappropriateanaestheticandanalgesicprotocolsisgenerallynotcompulsory.Inalargeproportionofcasesglobally,itiscurrentlyperformedbyfarmerswithoutanyanalgesiawhatsoever[1].Contributingtothisstateofaffairs,thereisacom-pletelackofmedicinesregisteredassafeandeffectiveforpainalleviationinthissettinginmanyjurisdictions.Internationallyharmonisedveterinarymedicineregulatoryrequire-mentsforthedevelopmentofsuchmedicinesrequirestudiesofthestand-alonesafetyimpactsofthemedications[10].Thecombineduseofsedation,injectedlocalanaesthesia,orsystemicanalgesiamayconfoundsystemicand/orlocaltoxicityfindingsduetothetopicalanaestheticsalone,andtherefore,arenotemployedinthissetting.2.2.Study1—TargetAnimalSafetyStudy—ExaminingSafetyofTri-Solfen®AdministrationtoCalfDisbuddingWoundsatupto5 theRecommendedDoseTheanimalphaseforStudy1wascompletedbetweenSeptemberandOctober2019attheUniversityofNewEnglandAnimalHouse,Armidale,Australia.Thirty-two(32)2–7-weeks-oldcalvesconsistingofsixteenentiremaleandsixteenfemaleanimalsfromapopulationofapproximately50mixed-sexdairycrossbredcalveswithconfirmedhornbudswereselectedforthisstudy.Theanimalswerestratifiedbysexandrankedfromheaviesttolightestonbodyweight(recordedonday 4).Theywerethensequentiallyblockedintofoursandrandomlyallocatedtofourtreatmentgroups(placebo,1 ,3 ,5 Tri-Solfen®treatment)fromwithineachblockviarandomnumberdraw.ThetreatmentregimeforanimalsinStudy1isoutlinedinTable1.Allcalvesweredisbuddedusingahotiron(18mm,KerblElectric,ShoofInternationalPtyLtd.,Cambridge,NewZealand).Followinganimalrestraint,theheatedhotironwasappliedoverthehornbudandrolledaroundthehornbudinacircularfashionseveraltimes,suchthataringoftissuearoundthebudwascauterisedthroughthefullthicknessoftheskin.Cauterisedhornbudtissue(~20mmdiameterassociatedwitheachbud)wasremovedwithforceps,providingamaximumabsorptivebedofwoundedtissue.Calvesweretreatedfollowingdisbudding,allowingfor30secforcauterisedtissuetocool.ThethreeTri-Solfen®treatmentgroupsthenreceiveddosagesat1 ,3 or5 themaximumrecommendeddoseof2mLperdisbuddingwound.Toavoidlargedosesresultinginexcessiverun-offofthetopicalanaestheticsolution,3 and5 dosageswereadministeredinrepeated2mLaliquotsapproximately1hraparttoeachdisbuddingwound.Alldosesweredeliveredwithinthe1–6hTmaxtimeframereportedforplasmaandtissuesofdisbuddedcalvesfollowinglidocaineandbupivacainetreatments[15].Additionally,allgroupswerere-treatedondays1and2approximately24hand48hfollowingtheirinitialtreatment,thusmeet-ingGL43guidelinesrequiringconsiderationofanextendeddurationofexposure[10].Eachdisbuddingwoundinplaceboanimalswastreatedwith2mLofa0.9%sterilesalinesolutioncontaining1%bluefoodcolouringtofacilitateblinding.Fromday 4,calveswerehousedinthesamebarninindividualadjacentpenseachwithwoodenslatflooring.Calvesneachtreatmentgroupwerepennedadjacenttooneanotherwithanemptypenbetweeneachtreatmentgroup.Calves’dietswereasperDairyAustraliaacceptedbestpractice.Thelevelofroughageonofferfollowedcurrentacceptedbestpractice(https://www.dairyaustralia.com.au/en/animal-management-and-milk-quality/calf-rearing;accessedon18March2021).Calveshadadlibitumaccesstopotablewater.Halfoftheanimalsineachgroupwereeuthanisedonday3,withtheremaindereuthanisedonday4.

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Animals2021,11,869 5of17 Table1.TreatmentregimeforanimalsinStudy1. GroupAnimals(n)TreatmentDoselevelDosingRegime 18Placebo-2mLsterilesalineappliedonDays0,1and2(2mLperdaytotal)28Tri-Solfen®1 2mLTri-Solfen®applieddailyonDays0,1and2(2mLperdaytotal)38Tri-Solfen®3 2mLTri-Solfen®appliedthreetimesat1hintervalsonDays0,1and2(6mLperdaytotal)48Tri-Solfen®5 2mLTri-Solfen®appliedfivetimesat1hintervalsonDays0,1and2(10mLperdaytotal)

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fen®appliedthreetimesat1hintervalsonDays0,1and2(6mLperdaytotal)48Tri-Solfen®5 2mLTri-Solfen®appliedfivetimesat1hintervalsonDays0,1and2(10mLperdaytotal) 2.2.1.AnimalObservationsIncludingWoundAssessmentStudyanimalswereobservedforthefirst30minfollowingeachtreatmentandthencehourly( 15min)foraminimumof6hfollowingthefinaltreatmentofeachgrouponeachday(0,1and2).Twicedailyobservations,undertakenbyablindedveterinarianduringthemorning,continuedpriortoeuthanasiaondays3or4.Observationsincluded:generalbehaviouranddemeanour,evaluationofanyappetitechange,ambulation,faecalconsistencyandcolour,skincondition,ocularandnasaldischarge,andanyneurologicorcardiorespiratorysignsthatmaybeindicativeofanadversedrugreaction.Bodyweightsofanimalsweremeasuredatday 4,andpriortoeuthanasia.Dailyfeedandwaterintakewerealsoassessedonanindividualanimalbasisoncedaily.Duringeachveterinaryexamination,disbuddingwoundswereexaminedforthepres-enceofoedema,erythema,discharge,alopeciaandflakingofskinontheareasurroundingthecauterisedwound.Inaddition,allapplicationsiteswerephotographedonthedayofnecropsy.2.2.2.BloodSamplingandAnalysisBloodspecimenswerecollectedfromthejugularveinofcarefullyrestrainedcalvesatthefollowingtimepoints:day 4,day0,andpriortoeuthanasia.Bloodsampleswerecollectedbyexperiencedveterinariansand/ortechniciansduringthemorning.Bloodwascollectedforcoagulation,biochemicalandhaematologicalanalysis.Serumandplasmawereextractedaftercentrifugation(ROTOFIX46,HettichAsiaPacificPty.Ltd,Singapore)at3500 rpmfor7minatambienttemperatureandstoredinindividual5mLvials.Athinbloodsmearwasalsoprepared.Specimens(serum,plasma,wholebloodinEDTAtubes)werethenstoredchilledandbloodsmearswerestoredatroomtemperatureandforwardedtothelaboratory(GribblesVeterinary,Christchurch,NewZealand)foranal-ysis.Standardbiochemical,haematologicalandcoagulationparametersweresubsequentlyassessedinbloodsamplescollectedatday 4,0andthedayofnecropsyandcomparedtopublishedreferenceranges[47–50].2.2.3.GrossPathologyandHistopathologyNecropsywasperformedonallanimalsbyblindedandexperiencedveterinarians.Follow-ingvisualexamination,representativesectionsofalltissues(e.g.,heart,brain,lung,liver,kidney,adrenalgland,applicationsitetissuefromtheedgeofthedisbuddingwound)werecollectedinto10%formalsalinesolutionandstoredforsubsequenthistopathologicalexamination.Sectionsoftissuesunderwenthematoxylinandeosinstaining.RepresentativesectionsofalltissueswerethenexaminedfromanimalsinGroups1–4.Additionally,histopathologywasevaluatedforanyorganwithagrossabnormalityfromanyanimal.Thecompletelistoftissuesincludedapplicationsiteskin,pituitarygland,thyroidgland,parathyroidgland,adrenalgland,pancreas,spleen,ovaries,uterus,testes,prostate,epididymis,heart,brain,brainstem,spinalcord,eye,lung,muscle,mammarygland,liver,gallbladder,kidney,urinarybladder,lymphnode,skin,boneandmarrow,marrowsmear,stomach,duodenum,jejunum,ileum,colon,caecumandthymus.Evidenceofpathologyand/orthepresenceof

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Animals2021,11,869 6of17

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Animals2021,11,869 6of17 multi-focalsurfacebacterialcolonisationwasscoredasfollows:0=normal,1=minimal,2=mild,3=moderate,4=marked.2.2.4.UrinalysisUrinespecimens(upto~5mL)werecollectedandrecordedfromallanimalsatnecropsyondays3and4usingasterileneedleandsyringedirectlyfromthebladderatautopsy.Detailedlaboratoryurinalysiswasperformedassessingarangeofparametersincludingbilirubin,colourandclarity,glucose,ketones,pH,protein,specificgravity,uro-bilinogen,presenceofblood(erythrocytesandhaemoglobin)andmicroscopicexaminationofsediment.2.2.5.StatisticalAnalysesKeyclinicalparameters,includingheartrate,respirationrate,rectaltemperature,bodyweightandbodyweightchange,aswellaskeybiochemical,haematological,urinaryandcoagulationparameters,werecomparedusingSASversion9.4(SASInstitute,Cary,NC,USA).Comparisonsbetweengroupswereperformedusingcovariancepatternrepeatedmeasuresmodelsandnon-parametrictests,withtreatmentandtimepointandothervariablesassessed.Datafromdays3and4inStudy1werecombinedintoasingletimepoint(“SacrificeDay”).Whereavailable,pre-treatmentdatawereincludedasaco-variate.Baselinedataweredefinedasthelatestmeasurementofagivenvariablebeforetreatmentwasadministered.Thestatisticalmodelincludedtreatment,sex(exceptforparametersonlymeasuredinonesex),andtreatment-by-sexinteractionsasfixedeffects,whiletherepeatedmeasuresANOVAmodelincludedtreatment,sexandtime(andtheir2-wayand3-wayinterac-tions)asfixedeffects.Datawerecomparedeitherwithinoracrosssexdependingonobservedinteractions.Modelswereselectedusingbackwardseliminationwhere3-wayand2-wayinteractiontermswiththehighestp-Valueweresequentiallydroppedfromthemodel.Theprocesswasperformedhierarchicallystartingwiththe3-wayinteractiontermsusingathresholdp-Valueof0.1,asrequiredbyregulatoryguidelines.Modelselectionwasstoppedwhenanyofthefollowingoccurred:(i)allinteractiontermsatthehighestlevelhadap-Value<0.1;(ii)nointeractiontermsremainedinthemodel.Thestatisticalmodelwastherefore:Parameter=Baseline_variables+Treatment+Sex+Time+Treatment:Sex+Treat-ment:Time+Sex:Time+Treatment:Sex:Time+Residual_ErrorLeastsquaresmeanswerecomparedatasignificancelevelofp<0.1inthefirstinstanceapartfromtreatmentbysexbytimepoint,whichwascomparedatp<0.05inthefirstinstance.Fortherepeatedmeasuresmodels,mostresidualerrorpatternswereselectedbasedonacomparisonofAkaikeInformationCriteria(AIC)outputformodelscontaininganunstructured(UN),autoregressive(AR;1),variancecomponents(VC)andindependentresidualerrorpattern(withthelowestAICpreferredamongthepossiblecovariancematrices).ThedenominatordegreesoffreedominthecovariancepatternrepeatedmeasuresmodelswerecalculatedusingtheSatterthwaiteapproximation.Modelsuitabilitywasas-sessedbyexaminationofresidualplots,preliminarydataexplorationfornormalityornon-normality,testsfornormalityandresultsfromhomogeneityofvariancetests.DatawerenottransformedwiththeexceptionofCreatineKinasewhichwaslog-transformed.Categoricalurinalysisdatawerecompared(proportionsofanimals/groupineachcategory)usingFisher’sExactTestacrosssex.Urinalysisdatareportedonanumericalscale(pHandspecificgravity)werecomparedbyKruskal–Wallistestwithexactp-ValuesestimatedusingMonteCarloestimation.

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Animals2021,11,869 7of17

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Animals2021,11,869 7of17 2.3.Study2—SafetyandEfficacyofTri-Solfen®AdministrationtoCalfDisbuddingWoundsunderFieldConditionsAnimalexperimentsforStudy2werecompletedbetweenNovemberandDecember2019oncommercialdairyfarmsinGloucester,NewSouthWales,Australia.Seventy-four(74)youngfemaleHolstein-FriesianandJerseycalves2–6weeksofagewereselectedfromtwosimilardairyherdslocatedwithin20kmofeachother.Calveswereidentifiedassuit-ableforselectionbasedonconfirmedoverallgoodhealthandpresenceofnormal,healthyhornbuds.Animalswereweighed,stratifiedbyweightandherdoforiginandrandomlyallocatedintotwotreatmentgroups—placebo(n=36)andTri-Solfen®(n=38)—using“drawfromahat”methodology.Priortodisbuddingandtreatment,allcalveswereexam-inedforsafetyassessmentsandsham-treated.Shamtreatmentinvolvedrestrainingandhandlingasperdisbuddingbutwithoutactualdisbuddingortreatmentandsubsequentalgometer(ForceCellModule50 0.02lbf;WagnerInstruments,Greenwich,CT,USA)andpainscoringonday 2andday 1.AllcalvesweredisbuddedusingahotironasdescribedinStudy1.Followingdisbudding,aperiodofapproximately30secwasallowedforthetissuetocoolbeforeapplicationoftheplacebo(1 2mL0.9%salinesolutionwithbluefooddye)orTri-Solfen®(1 2mLTri-Solfen®)solution.Trialcalveswerehousedincoveredpens(multiplecalves/pen)forthedurationofthestudy.Calveswereprovidedwithadlibitumaccesstopotablewater,pelletsandroughageasperbestpracticeatthetrialsites.Studyanimalswereretainedbytheirherdoforiginattheconclusionofthein-lifephaseofthestudyatday33–34.2.3.1.ClinicalExaminationDetailedindividualclinicalexaminationswereperformedonselectedcalvesonday 2andday 1followingsham-treatment.Clinicalexaminationswerethenrepeatedimmediatelypriortopainassessmentsat22h,days7–8anddays11–12.Examinedpa-rametersincludedgaitandactivity,eating/drinking,urination/defecationandgeneralcalfdemeanour.Disbuddingwoundswerevisuallyexaminedondays3–4,days7–8,days21–22anddays33–34.Disbuddingwoundswereassessedforoverallappearance,woundresolutionandthepresence/absenceofinfectionorotheradverseclinicalfindings.Woundswereclassifiedaseither“normal”or“abnormal”.Aclassificationofanormalwoundwasdefinedasthepresenceofadrywoundwithepithelialcontraction.Anabnor-malwoundwasdefinedasawoundwiththepresenceof(i)anopenwound;and/or(ii)necrotictissue;and/or(iii)thepresenceofsignificantpus.Calveswerealsoweighedondays 2,days11–12,days21–22anddays33–34usingelectronicstockscales(Ruddweigh300,Gallagher,Australia).Averagedailygainwascalculatedonanindividualanimalbasisovertheperiodspre-proceduretodays11–12,days21–22anddays33–34.EfficacymeasurementswereinitiallyplannedasapartofStudy2,includingwoundsensitivitytestingusingahand-heldalgometer(ForceCellModule50 0.02lbf;Wag-nerInstruments)andvideorecordingofpain-relatedbehaviourincludingearflicksandheadshakes.Althoughinitiallyrecorded,analysiswasnotprogressedasextremeenvi-ronmentalconditions,includingwavesofflyinfestationoccurred,andwerefelttohavelikelyconfoundedandcompromisedthesensitivityandspecificityoftheproposedefficacymeasurements.2.3.2.StatisticalAnalysesSafetyassessmentparameterdata(bodyweights,disbuddingwounddataandclinicalexaminationdata)wereenteredintoMicrosoftExcel(Microsoft,Redmond,WA,USA).AllstatisticalcomparisonswereperformedusingStatistix10.0(AnalyticalSoftware).Aver-agedailygainwascalculatedusingindividualanimalweightsattherelevanttimepointsandtheelapsedtimebetweenpointsforeachanimal.Parameterdatawerecompared

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Animals2021,11,869 8of17 betweengroupsusingRepeated-MeasuresAnalysisofVarianceandamodelthatincluded“Time”asawithin-subjectfactor.Treatment,timeandtreatment timewerecomparedusingTukey’sAllPairwiseComparisonTestatp<0.05.AveragedailygainwascomparedbetweentreatmentsfortherelevanttimeperiodsusingAnalysisofVarianceandamodelthatincluded“Site”.Meanswereagaincomparedatp<0.05.Proportionsofnormalandabnormaldisbuddingwoundsatdays7–8and11–12werecomparedbetweengroupsusingFisher’sExactTestatp<0.05.3.Results3.1.Study1—TargetAnimalSafetyStudy—SafetyofTri-Solfen®AdministrationtoCalfDisbuddingWoundsIncludingupto5 theRecommendedDose3.1.1.ClinicalExaminationsAnimalsremainedvisiblywellthroughouttheexperimentalperiod.Therewerenorecordedseriousadverseevents,andnomortalityofcalvesduringthestudyperiod.Groupmeanvalues(TableS1forthekeyclinicalparameters,includingrectaltemperature,heartrate,respirationrate,waterintake,andfeedintake)betweenGroup1andthetreatmentgroups(Groups2–4)weresimilarovertime(Table2)withnoevidenceofadose–titrationeffect.Bodyweightsandaveragedailygainwerealsosimilarwithnostatisticallysignificantdifferencesobservedbetweengroups(Table2).Table2.Statisticalanalysisofkeysafetyparametersandobservedp-Valuesfortreatment(andtimeasappropriate).Statisticallysignificantresultsareindicatedinbold. Parameterp-Value(Treatment)p-Value(Time) Bodyweight0.117<0.001RectalTemperature0.7270.482HeartRate0.443<0.001RespirationRate0.601<0.001AverageDailyGain0.126- WaterIntake0.998<0.001FeedIntake0.026<0.001

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0.117<0.001RectalTemperature0.7270.482HeartRate0.443<0.001RespirationRate0.601<0.001AverageDailyGain0.126- WaterIntake0.998<0.001FeedIntake0.026<0.001 RedBloodCells0.0030.081Haemoglobin0.0040.112Haematocrit0.0030.003WhiteBloodCells0.3200.342MeanCorpuscularVolume0.596<0.001MeanCorpuscularHaemoglobin0.5180.635MeanCorpuscularHaemoglobinConcentration0.4300.002ActivatedPartialThromboplastinTime0.0560.500ProthrombinTime0.0560.166Fibrinogen0.1680.000 Alanineaminotransferase0.1510.138Albumin0.4310.000Alkalinephosphatase0.595<0.001Aspartateaminotransferase0.943<0.001Creatinine0.225<0.001Log2CreatineKinase0.8290.124Gamma-glutamyltransferase0.4680.006Globulin0.0480.000LactateDehydrogenase0.2300.001Totalprotein0.5960.169Urea0.6310.247

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Animals2021,11,869 9of17 Table2.Cont. Parameterp-Value(Treatment)p-Value(Time) Colour0.886-Turbidity1.000-Blood0.010-Protein0.832-Bilirubin1-RedBloodCells0.895-WhiteBloodCells0.126-UnidentifiedCrystallineStructures0.886-BilirubinCrystals0.886-AmorphousUrateCrystals0.587-StruviteCrystals0.893-EpithelialCells0.587-AmorphousDebris0.073-pH0.060-SpecificGravity0.569-

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s0.886-BilirubinCrystals0.886-AmorphousUrateCrystals0.587-StruviteCrystals0.893-EpithelialCells0.587-AmorphousDebris0.073-pH0.060-SpecificGravity0.569- 3.1.2.HaematologicalandUrineAnalysisHaematologicalanalysisdetectedasmallbutstatisticallysignificantdifferenceforRedBloodCells,HaemoglobinandHaemocrit,betweenGroup1(placebo)andGroup3(5 dosegroup);however,groupmeansremainedintheuppernormalofthereferencerangeforthedurationofthestudy(TableS2).Noothersignificantdifferencesinhaematologicalparam-eters(MeanCorpuscularVolume,MeanCorpuscularHaemoglobin,MeanCorpuscularHaemoglobinConcentration,WhiteBloodCells,ActivatedPartialThromboplastinTime,ProthrombinTimeandFibrinogen)wereobserved.Groupmeanvaluesforthekeybiochemicalparameters(AlanineAminotransferase,Albumin,AlkalinePhosphatase,AspartateAminotransferase,Creatinine,CreatineKi-nase,Gamma-Glutamyltransferase,Globulin,LactateDehydrogenase,TotalProteinandUrea)followedsimilartrendsovertimewithanydifferencesfoundbetweengroupsnotstatisticallysignificant(TableS3).Nodivergencebetweenparameterswasobservedathigherdoses.Urinespecimensfromanimalsineachgroupwereanalysedforarangeofkeyparam-eters.Asignificantdifferencewasnotedfordetectableblood(basedonpseudoperoxidaseactivityofhaemoglobinandmyoglobin)betweenGroup4(5 dose)andGroup1(placebo)(p<0.010).Nosignificantdifferenceswereobservedforanyotherparameters(Table2).3.1.3.GrossPathologyofAnimalsFollowingEuthanasiaAllanimalswereeuthanisedandsubjectedtonecropsyatdays3and4followingdisbuddingandtreatment.Ongrossexaminationatnecropsy,lungconsolidationwasobservedin10/32(31.3%)ofanimals,consistentwiththepresenceofcurrentorhistoricalsub-clinicalpneumonia.Ofthe10animals,3(30%)werefromtheplacebogroup,2(20%)werefromGroup2,3(30%)werefromGroup3and2(20%)werefromGroup4.Nootherlesionsorabnormalitieswereobservedwithanyotherorgansystem.3.1.4.HistopathologicalAnalysisofTissueSamples,IncludingSkinHistopathologicalanalysisoftissuesampleswasundertakenonallGroup1(placebo)andGroup4(5 dose)animals.Noevidenceofpathologywasnotedinanyorgansampleexamined.ThemicroscopicfindingsfromhistopathologicalanalysisofskinsamplesfromthewoundsitearepresentedinTable3.Locallyextensiveordiffusecutaneousnecrosiswithhaemorrhage,oedema,crusting,mineralisation,inflammatoryinfiltratesandfibrosisweresimilarinallcalvesacrossGroups1–4.Therewasanoticeablereductioninthequantity

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Animals2021,11,869 10of17 ofsurface-localisedbacteria,gradedusingasubjectivescalebetweenGroups2,3and4(Tri-Solfen®treatmentgroups)andGroup1,theplacebogroup.Table3.Incidenceofpathologicalconditionsand/orthepresenceofmulti-focalsurfacebacterialcoloniesinskinsamplescollectedfromanimalsinStudy1. FindingaGroup1(n=8)Group2(n=8)Group3(n=8)Group4(n=8) Locallyextensiveordiffuseepidermalcoagulativenecrosiswithhaemorrhage,oedema,neutrophilicinfiltrates,serumcrusting,occasionalmineralisation----0000010000200003000048888 Surfacebacterialcolonies,multifocal----0000111457220103130044120 Dermalperivascularlymphoplasmacytic,neutrophilicandeosinophilicinfiltrates,multifocal----0000010000256743321440000 Dermalfibrosis----0000012104225613422340000

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perivascularlymphoplasmacytic,neutrophilicandeosinophilicinfiltrates,multifocal----0000010000256743321440000 Dermalfibrosis----0000012104225613422340000 aGradingscale;0=normal,1=minimal,2=mild,3=moderate,4=marked.3.2.Study2:SafetyandEfficacyofTri-SolfenAdministrationtoCalfDisbuddingWoundsunderFieldConditions3.2.1.EnvironmentalConditionsArangeofextremeenvironmentalconditionsoccurredduringthestudyperiod,includinganextendedheatwavewithdaytimetemperaturesconsistently>40 C.Catas-trophicbushfireconditionsweredeclaredbytheAustralianBureauofMeteorologyforthestudyarea.Significantairpollutionsecondarytobushfiresmokeandwavesofflyinfestationwerealsoexperiencedduringthestudyobservationperiod.3.2.2.ClinicalExaminationsDetailedindividualclinicalexaminationsbyaveterinarianwereperformedonallselectedcalvesonday 2andday 1(followingshamdehorning)andimmediatelypriortopainassessmentsat22hpostprocedureandondays7–8anddays11–12.Examinationparametersincludedheartrate,respirationrate,rectaltemperature,gastrointestinalactivity,gaitandlocomotion.Therewasatrendtowardshigherrectaltemperaturesinplacebotreatedanimals.Meanrectaltemperatureswerehigherintheplacebotreatmentgroupatalltimepointsexceptdays7–8.Themostmarkeddifferencesoccurredonday1whengroupmeanrectaltemperaturesof39.5 Cand39.1 Cwereobservedforplacebo-treatedand

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Animals2021,11,869 11of17 Tri-Solfen®-treatedcalves,respectively(p=0.06).Nosignificantdifferenceswereobservedbetweentreatmentgroupsforheartrate(p=0.53)orrespirationrate(p=0.64)(Table4).Table4.Meanclinicaldata(rectaltemperature,heartrate,respiratoryrate)forPlaceboandTri-Solfen®-treatedcalvesovertimeinStudy2. Group/TreatmentRectalTemperature( C)HeartRate(beats/min)RespiratoryRate(breaths/min) Day 2Placebo38.8 0.49128.9 19.344.0 8.1Tri-Solfen®38.7 0.53128.4 22.747.0 10.6 Day 1Placebo38.8 0.30129.3 20.041.5 9.2Tri-Solfen®38.7 0.36128.5 26.240.4 11.0Day1---Placebo39.5 0.50125.1 14.644.5 10.2 Tri-Solfen®39.1 0.46131.0 20.743.7 9.7 Day7–8Placebo39.9 0.52117.1 21.351.7 19.5Tri-Solfen®39.9 0.55121.1 27.250.7 17.8 Day11–12Placebo39.3 0.51123.8 20.744.2 10.9Tri-Solfen®39.1 0.55124.7 19.443.1 10.9 Calveswereweighedonday 2,days11–12,21–22,and33–34andaveragedailygainwascalculatedonanindividualanimalbasisovertheassessmentperiod.TheseresultsarepresentedinTable5.TherewasatrendtowardshigheraveragedailygaininTri-Solfen®-treatedanimals.AveragedailygainswerehigherintheTri-Solfen®treatmentgroupatalltimepoints,whichwasparticularlynotableatdays11–12(p=0.06).Table5.Averagedailygain(kg/day)incalvesfromeachtreatmentgroupovertimeforStudy2. Group/TreatmentAverageDailyGain(Day 2toDay11–12)kg/dayAverageDailyGain(Day 2toDay21–22)kg/dayAverageDailyGain(Day 2toDay33–34)kg/day Placebo0.650.810.83Tri-Solfen®0.850.890.88Treatmenteffect(AverageDailyGain)0.200.080.05Treatmenteffect(%)31106

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AverageDailyGain(Day 2toDay33–34)kg/day Placebo0.650.810.83Tri-Solfen®0.850.890.88Treatmenteffect(AverageDailyGain)0.200.080.05Treatmenteffect(%)31106 OneanimalintheTri-Solfen®treatmentgroupwasfounddeadinitspenonthemorn-ingofday1.Asmallamountofconsolidatedlungtissuewasobservedpostmortem,sug-gestiveofviralpneumonia,withnootherabnormalitiesdetectedatnecroscopy.Nootherseriousadverseeventsormortalityoccurredduringthestudy.3.2.3.WoundHealingAssessmentDisbuddingwoundswerevisuallyexaminedondays7–8,11–12,21–22and33–34andassessedforoverallappearance,woundresolutionandthepresence/absenceofinfectionorotheradverseclinicalfindings.ProportionsofnormalandabnormaldisbuddingwoundsateachtimepointarepresentedinTable6.Relativelyhighproportionsofwoundswereclassifiedasabnormalatdays7–8,(56%and46%inplaceboandTri-Solfen®treatedcalves,respectively).Whileasimilarratio(50%)ofabnormalwoundswerestillpresentintheplacebogroupatdays11–12,thisnumberwassignificantlyreducedto20%(p<0.05)inthe

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Animals2021,11,869 12of17 Tri-Solfen®treatmentgroupatthistimepoint.Thedifferencesbetweentreatmentgroupsdisappearedbydays21–22withwoundscontinuingtohealnormallybyday33–34.Table6.ProportionsofnormalversusabnormaldisbuddingwoundsinanimalsbetweentreatmentgroupsandovertimeforStudy2. TimepointTreatmentGroupAbnormal(%)Normal(%) Days7–8Placebo(n=36)20(55.6)16(44.4)Tri-Solfen®(n=35)16(45.7)19(54.3)Days11–12Placebo(n=34)17(50.0)17(50.0)Tri-Solfen®(n=35)7(20.0)28(80.0)Days21–22Placebo(n=36)3(8.3)33(91.7)Tri-Solfen®(n=36)3(8.3)33(91.7)Days33–34Placebo(n=36)0(0.0)36(100)Tri-Solfen®(n=36)0(0.0)36(100)

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35)7(20.0)28(80.0)Days21–22Placebo(n=36)3(8.3)33(91.7)Tri-Solfen®(n=36)3(8.3)33(91.7)Days33–34Placebo(n=36)0(0.0)36(100)Tri-Solfen®(n=36)0(0.0)36(100) 4.DiscussionThereisanurgentimperativetodevelopveterinarymedicinesregisteredassafeandeffectiveforusetoalleviatepainininfantlivestockundergoingroutinehusbandryprocedures,includingdairycalfdisbudding.Applieddirectlytothedisbuddingwound,Tri-Solfen®isreportedtobeeffectivetomitigatepost-operativepain[7,8].Toachieveregulatoryapprovalforsuchuses,analgesicmedicinesmustmeethighstandardsofproofofsafety,requiringdetailedproscribed“marginofsafety”studiesexaminingthetoxiceffectsofdrugsatrecommendeddoseandpotentialoverdoselevels.Thesetrials,performedtointernationallyharmonizedstandards,areneverthelessinvasiveandmayinvolveethicalconstraintssuchastherequirementforsingleproductuse,where,innon-regulatorytrials,multi-modalpaintherapymaybeusedforoptimalwelfare.Theneedforsuchtrialsisreducedhowever,whereversufficienthigh-qualitydataarepubliclyavailable.Lidocaineandbupivacaine,thelocalanaestheticsinTri-Solfen®,althoughwidelyusedinhumanandveterinarymedicineformanydecades,arenotregisteredforusetomitigatepainincalvesinmostjurisdictions.Althoughtheiracutesystemicneuro-andcardiotoxiceffectsarewelldescribed,unfortunately,atthetimeofthesetrials,therewasinsufficientinformationavailabletomeetregulatoryrequirementsforproofofsafetyofuseincalves,includinglackofinformationregardingpotentialwiderbiochemical,haematologicalortissuetoxiceffects,orimpactswhenusedviatopicalapplicationtosignificantopenwounds.WethereforehaveperformedregulatoryrequiredmarginofsafetystudieswithTri-Solfen®andreporttheoutcomeofthesetocontributetothepublicrecord.Itishopedthatthesedatamayreducetheneedforsuchtrialsforregistrationoflidocaineorbupivacainecontainingproductsforcalvesinthefuture.WeseparatelyinvestigatedandreportedlocalanaestheticpharmacokineticdataincalvesfollowingtopicalapplicationofTri-Solfen®tothedisbuddingwoundattherecommendeddose(2mLperhornbud)[15].Inthecurrentstudies,wereportalackofevidenceofsignificanttoxiceffectsornegativeimpactonwoundhealingfollowingTri-Solfen®useoncalfdisbuddingwoundsatuptofivetimestherecommendeddose.Converselyatrendtowardsreducedbacterialloadandimprovedhealingwasevidentinthefirst4–14daysfollowingtreatment,respectively.NoevidenceofsignificanttoxicitywasobservedinTri-Solfen®-treatedanimals,withgeneralclinical,biochemicalandhistopathologicaldatafromanimalsintheplaceboandTri-Solfen®treatmentgroupssimilarandremainingwithinnormalparametersdespitereceivingupto5 therecommendeddose.Wedidnoteaminor,butstatisticallysignificantdifferenceintheamountofmicroscopicblood(measuredbydetectionofhaemoglobinormyoglobinbydipsticktest)detectedinthe5 dosegroupcomparedtotheplacebogroupanimals.Thiswasnotassociatedwithanincreaseinredbloodcellsintheurine,andtherewasnoevidenceofabnormalitiesdetectedonhistopathologicalanalysisoftissuesamplescollectedfromthekidneys,uretersandbladdertosuggestanydirecttoxiceffectstoanyofthesetissues.Thefindingofhaemoglobin/myoglobininurinewithoutanincreaseinred

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Animals2021,11,869 13of17

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Animals2021,11,869 13of17 bloodcellscouldrelatetothedegreeoftissuetraumaand/orhaemolysisatthewoundsite.Cauterisationproceduresresultinmuscletissueandredbloodcelldamage,releasingmyoglobinandhaemoglobin,respectively,whichmaybesubsequentlyreabsorbedandeliminatedintheurine.Woundsize/depthwasnotmeasuredasacorrelateinthisstudytoassesswhetherdifferencesmayhaveexistedbetweengroups.Histopathologicalanalysisdidnotnoteanydifferenceintissuetraumabetweengroupsatthewoundsite;however,thismaynotbesensitivetominordifferencesinredbloodcelldamageatthetimeoftheprocedureorafter.Therearereportstosuggestthathighconcentrationsoflidocaineapplieddirectlytoredbloodcellsmayinducehaemolysis[51].Itispossiblethereforethatincreasedbreakdownofredbloodcellscontainedincrustatthewoundsiteoccurredinthe5 treatmentgroup,ascomparedwiththeplacebogroup,contributingtothisfinding.Intravascularhaemolysisisanotherpotentialexplanation.Althoughnot(toourknowl-edge)reportedinassociationwithbupivacaineadministration,rarecasesofintravascularhaemolysishavebeenreportedinassociationwithlidocaineadministration[52].Typically,however,thisisonlyinthesettingofacutetoxicitywithotherfactorspredisposingtooxidativestressandmethaemoglobinaemia[52].Ontheotherhand,otherreportshavedocumentedthatlidocaineprovidesaprotectiveeffectagainstintravascularhaemoly-sis[53].Therewasalowgradebutstatisticallysignificantreductioninredbloodcellsandhaemoglobininbloodinanimalsinthe5 dosegroup(Group4)comparedtotheplacebo(Group1)group;however,haemoglobinandredbloodcellvaluesinbothgroupsremainedwellwithinthenormalrangeforcalves,andmeancorpuscularvolumeandhaemoglobinconcentrationwerealsonotsignificantlydifferentbetweengroups.Thissuggeststhat,ifintravascularhaemolysiswaspresentinthe5 overdosegroup,itwasofaverylowgradeandinsufficienttohaveasignificantnegativeimpactonanimalhealth.WealsofoundnoevidenceofnegativeimpactsonanimalhealthfollowingTri-Solfen®treatmentunderfieldconditionsinStudy2,despitetheunanticipatedextremeenviron-mentalconditionsthatoccurredoverthecourseofthestudy.OneanimalintheTri-Solfen®-treatedgroupwasfoundtohavedied24hfollowingtreatment;however,thiswasconsideredunlikelytoberelatedtotreatmentastherewerenoclinicalsignsoftoxicityatearliertimepointssuchasintheearlyminutesandupto4–6hpost-administrationwhenlocalanaestheticconcentrationswerelikelytohavepeaked[15],suchastosuggestacutelocalanaestheticsystemictoxicityorararehypersensitivityreaction[54,55].Further-more,theonlyabnormalitydetectedatnecroscopywaspneumonia,andcalfsurvivalwithpneumoniamayhavebeencompromisedbytheextremeenvironmentalconditions.Thesedataarethusconsideredtobeconsistentwithpreviousdatareportedinpiglets,cattleandsheep,showinganabsenceoflocalorsystemictoxiceffectsofTri-Solfen®treatmentatrecommendeddosesduringand/oraftersurgicalhusbandryprocedures[9,28,38,41,42].Theyarealsoconsistentwithpreviousstudies,limitedinnumber,identifyinganabsenceoflocalanaesthetic-relatedtoxiceffectsonhaematological,biochemicaland/ortissuesparameters,whenappliedatdosesthatdonotinduceCNSorCVStoxicity[15,56–59].Collectively,thesedataarethusconsideredtosupporttheconclusionthatthelocalanaes-thetics,asformulatedinTri-Solfen®,areminimallyabsorbedacrossthethermocauterydisbuddingwoundincalves,anddonotgenerateacutetoxiceffects,despiteadministrationtoupto5 themaximumrecommendeddose.Intermsoflocalapplicationsitetoxicity,ourresultsarealsoconsistentwithpreviouspublisheddatasuggestinglittlenegativeimpactoflidocaineandbupivacainetopicalappli-cationtoopenwounds,whenusedinconcentrationscommensuratewiththosepresentinTri-Solfen®[21–29].Inourtargetanimalsafetytrial(Study1),despitetheapplicationofdosesofTri-Solfen®significantlygreaterthantherecommendeddose,nodifferencesinwoundhealingwereobservedascomparedwithplacebo-treatedanimalsoverthedura-tionofthetrial.Thesegrossobservationswereconfirmedbyhistopathologicalanalysisofwoundbiopsieswithextensivetissuenecrosisobservedinallanimals,regardlessoftreatment,indicativeofdamagecausedbythedisbuddingprocedureitselfandnoevidenceofanyadditionaladverseeffecttriggeredbytheapplicationoflocalanaestheticssuchas

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Animals2021,11,869 14of17

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Animals2021,11,869 14of17 secondarytocytotoxicity.ThislackofevidenceforanydeleteriouscytotoxicactivityinTri-Solfen®-treatedanimalswasconfirmedinourfieldtrialswithdisbuddingwoundsinplaceboandalltreatmentgroupsshowingsimilarhealingatday33–34followingtreat-ment.TheseresultsarealsoconsistentwiththerecentworkofStilwellandLaven[40],whichfailedtofindanydeleteriouseffectonwoundhealinginTri-Solfen®-treatedani-malsoneweekafterdisbuddingandtreatmentascomparedwithanimalstreatedwithtopicaltetracycline.Whilenodeleteriouseffectsonwoundhealingwereobserved,bycontrast,therewasevidenceofimprovedwoundhealingoverthefirst1–2weeksfollowingtreatmentinTri-Solfen®-treatedanimals.Placebo-treatedcalveshadhighernumbersofabnormalwounds,attributedtowoundinfectionbyveterinaryinspection,onday11–12inthefieldtrial(Study2).ThiswasassociatedwithhigherrectaltemperaturesandreducedaveragedailygaininplaceboascomparedwithTri-Solfen®-treatedcalvesoverthesameperiod.Thisissuggestiveofincreasedinfectionratesandgreater“set-back”inplacebo-treatedanimalsoverthefirst1–2weeksfollowingtheprocedure.“Set-back”involvingreducedaveragedailygainiscommoninanimalpost-surgicalhusbandryproceduresandisattributedtoincreasedcatabolicrateand/orreducedfeedintakesecondarytopainandthesurgicalstressresponsetotissuetrauma[60].Thismaybeexacerbatedbywoundinfection.Thisistypicallyfollowedbya“catch-up”phaseasthewoundshealandthesurgicalstressresponseabates[61].ResultsfromourfieldtrialsuggestedTri-Solfen®treatmentcontributedtoimprovedearlywoundhealingandreducedset-backinthefirst1–2weeksfollowingtheprocedure.Theseresultsareconsistentwiththosereportedbyothers.Lomaxetal.similarlyreportedfasterratesofwoundhealinginTri-Solfen®-versusplacebo-treatedlambsinthefirst14daysfollowingsurgicalhusbandryproceduresforflystrikeprevention[28].Cuttanceetal.[7]reportedatendency(p=0.09)towardsimprovedaveragedailygainincalvesdisbuddedwithpre-operativesedation,andpost-proceduralTri-Solfen®treatment,ascomparedtothosedisbuddedwithouttreatment.VandeSaagetal.[62]reportedimprovedweightgainincalvescastratedanddisbuddedwhentreatedwithmeloxicamandTri-Solfen®,ascomparedwithuntreatedanimals.Itisnotknownwhethersucheffectsmayrelatetoreductionsinpain,reductionininflammationandthesurgicalstressresponse,and/orreductioninbacterialcolonisationandwoundinfectionrates,oracombinationofthethree.EvidenceforthepotentialbeneficialantimicrobialeffectofTri-Solfen®treatmentandapotentialexplanationfortheimprovedearlywoundhealingweobservedinourfieldtrialcanbeseeninourobservationsofmicrobialcolonisationofdisbuddingwoundsfollowinghistopathologicalanalysis.ReducedmicrobialcolonisationofthewoundswasevidentinTri-Solfen®ascomparedwithplacebo-treatedanimalsfromsamplescollectedatnecroscopy(i.e.,3–4daysfollowingtreatment).Similareffects(reducedpusdischargeandbacterialcolonycounts)havebeenreportedfollowinguseofcetrimidecontainingantisepticsonsur-gicalwoundsindogs[63],suggestingthatitislikelythatthiseffectisduetothepresenceoftheantiseptic,cetrimide,inthetopicalanaestheticformulation.Localanaestheticsalsopossesssomeantibacterialactivity[32,64]thatmayhavecontributedtoareductioninthemicrobialloadinthedisbuddingwound,and,hypothetically,thereducedriskofabnor-malwoundsweobservedatday11/12inStudy2.MoredetailedstudiestocharacterizethewoundhealingefficacyandantimicrobialactivityofTri-Solfen®treatmentfollowingdisbuddingarewarranted,particularlyifitcanbeshownthatTri-Solfen®treatmentcanreducetheincidenceofwoundinfectionssimilarlyorwithgreaterefficacythanthepro-phylacticuseoftopicalantimicrobialsprays.Thishaspotentialbeneficialeffectstoreducetheneedforantibioticusebothforprophylaxisand/ortotreatwoundinfections,thelatterantibiotictreatmentscontributingtotheemergenceofantimicrobialresistance[65].5.ConclusionsTheresultsofthesesafetystudiesrevealthatthetopicalapplicationoflidocaineandbupivacainewithadrenalineandcetrimide,aspresentinTri-Solfen®,todisbudding

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Animals2021,11,869 15of17 woundsincalvespresentsaminimalriskofacutelocalorsystemictoxicityevenatdosessignificantlyhigherthantherecommendeddosage.Atrecommendedandoverdoselevels,noevidencecouldbefoundforanadverseeffectonwoundhealing.Conversely,Tri-Solfen®treatmentmayhavebeneficialimpactsonwoundhealing,possiblysecondarytolowerbacterialcolonisationandtheincidenceofwoundinfections.SupplementaryMaterials:Thefollowingareavailableonlineathttps://www.mdpi.com/2076-2615/11/3/869/s1,TableS1:GroupmeankeyclinicalparametersovertimeforanimalsinExperiment1,TableS2:GroupmeankeyhaematologicalparametersovertimeforanimalsinExperiment1,TableS3:GroupmeankeybiochemicalparametersovertimeforanimalsinExperiment1.AuthorContributions:Conceptualization,M.S.,M.C.andB.S.;methodology,M.S.,M.C.andB.S.;formalanalysis,M.C.,B.S.;investigation,M.C.,B.S.;resources,M.C.andB.S.;datacuration,M.C.andB.S.;writing—originaldraftpreparation,A.P.;writing—reviewandediting,M.S.,A.P.,M.C.andB.S.;projectadministration;M.C.andB.S.;fundingacquisition,M.S.Allauthorshavereadandagreedtothepublishedversionofthemanuscript.Funding:FundingforthisstudywasprovidedbyAnimalEthicsPtyLtd.,YarraGlen,Victoria,NSW,Australia.InstitutionalReviewBoardStatement:ThestudywasdesignedinaccordancewithGoodClinicalPractice(GCP)andVICHtargetanimalsafetystudyguidancedocuments.EthicsapprovalswereprovidedbytheUniversityofNewEnglandAnimalEthicsCommittee,Armidale,Australia(19-078and19-095).DataAvailabilityStatement:Thedatapresentedinthisstudyareavailableathttps://www.mdpi.com/2076-2615/11/3/869/s1.Acknowledgments:WethankBruceChickforhisexpertassistancewiththisproject.ConflictsofInterest:ResearchwassponsoredbyAnimalEthicsPtyLtd.,andcarriedoutbyinde-pendentveterinaryresearchcompanyInvetusPtyLtd.,toGCPandVICHstandards,toaddressnationalandinternationalveterinarymedicinesregulatoryapprovalrequirements.M.SheilisaninventorofTri-Solfen®,aFoundingDirectorandindirectshareholderofAnimalEthicsPtyLtd.Thefundershadanoversightroleinthedesignofthestudytoensurecompliancewithrequiredstandards.Thefundershadnoroleinthecollection,analyses,orinterpretationofdataorinthewritingofthefinalstudyreport:Thefundersprovidedconsenttopublishthestudyresultsandcontributedtodraftingthemanuscriptfromthefinalstudyreport.References1.Gottardo,F.;Nalon,E.;Contiero,B.;Normando,S.;Dalvit,P.;Cozzi,G.Thedehorningofdairycalves:Practicesandopinionsof639farmers.J.DairySci.2011,94,5724–5734.[CrossRef]2.Grondahl-Nielsen,C.;Simonsen,H.B.;Lund,J.D.;Hesselholt,M.Behavioural,endocrineandcardiacresponsesinyoungcalvesundergoingdehorningwithoutandwithuseofsedationandanalgesia.Vet.J.1999,158,14–20.[CrossRef][PubMed]3.Graf,B.;Senn,M.Behaviouralandphysiologicalresponsesofcalvestodehorningbyheatcauterizationwithorwithoutlocalanaesthesia.Appl.Anim.Behav.Sci.1999,62,153–171.[CrossRef]4.Frahm,S.;DiGiminiani,P.;Stanitznig,A.;Schoiswohl,J.;Krametter-Frotscher,R.;Wittek,T.;Waiblinger,S.NociceptiveThresholdofCalvesandGoatKidsUndergoingInjectionofCloveOilorIsoeugenolforDisbudding.Animals2020,10,1228.[CrossRef]5.Faulkner,P.M.;Weary,D.M.Reducingpainafterdehorningindairycalves.J.DairySci.2000,83,2037–2041.[CrossRef]6.Stafford,K.J.;Mellor,D.J.Dehorninganddisbuddingdistressanditsalleviationincalves.Vet.J.2005,169,337–349.[CrossRef]7.Cuttance,E.L.;Mason,W.A.;Yang,D.A.;Laven,R.A.;McDermott,J.;Inglis,K.Effectsofatopicallyappliedanaestheticonthebehaviour,painsensitivityandweightgainofdairycalvesfollowingthermocauterydisbuddingwithalocalanaesthetic.N.Z.Vet.J.2019,67,295–305.[CrossRef][PubMed]8.Espinoza,C.;Lomax,S.;Windsor,P.Theeffectofatopicalanestheticonthesensitivityofcalfdehorningwounds.J.DairySci.2013,96,2894–2902.[CrossRef][PubMed]9.Lomax,S.;Windsor,P.A.Topicalanesthesiamitigatesthepainofcastrationinbeefcalves.J.Anim.Sci.2013,91,4945–4952.[CrossRef][PubMed]10.EuropeanMedicinesAgency.VICHTopicGL43—Step7—GuidelineonTargetAnimalSafetyforVeterinaryPharmaceuticalProducts;EuropeanMedicinesAgency:London,UK,2008.11.Mather,L.E.Theacutetoxicityoflocalanesthetics.ExpertOpin.DrugMetab.Toxicol.2010,6,1313–1332.[CrossRef]

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