13.5.83 OfficialJournaloftheEuropeanCommunities NoL126/23
COUNCILDIRECTIVE
of18April1983
onthefixingofguidelinesfortheassessmentofcertainproductsusedinanimalnutrition
(83/228/EEC)
Whereastheguidelineshavebeendrawnuponthebasis
ofpresentscientificandtechnicalknowledgeandthey
maybeadaptedifnecessarytoanydevelopmentsinthis
sphere,
HASADOPTEDTHISDIRECTIVE :
Article1
MemberStatesshallprescribethatthedossiersonthe
productslistedinpoints1.1and1.2oftheAnnexto
Directive82/471/EECaretobecompiledinaccordance
withtheguidelinessetoutintheAnnexhereto.
Article2
MemberStatesshallbringintoforcethelaws,regulations
oradministrativeprovisionsnecessaryinordertocomply
withthisDirectiveby13July1984atthelatest.They
shallforthwithinformtheCommissionthereof.THECOUNCILOFTHEEUROPEANCOMMUNITIES ,
HavingregardtotheTreatyestablishingtheEuropean
EconomicCommunity ,
HavingregardtoCouncilDirective82/471/EECof
30June1982concerningcertainproductsusedinanimal
nutrition(J),andinparticularArticle7thereof,
HavingregardtotheproposalfromtheCommission ,
WhereasDirective82/471/EECprovidesthatthe
productsbelongingtocertaingroupsmustbeexamined
onthebasisofadossierforwardedofficiallytothe
MemberStatesandtheCommission ;
Whereassuchdossiersmustmakeitpossibletoverifythat
theproductsinquestioncomplywiththegeneral
principleslaiddownintheDirectiveinrespectofthe
inclusionofnewproductsintheAnnex;
Whereasithasbeenfoundnecessarytoprovideforthe
dossierstobecompiledinaccordancewithcommon
guidelinesdefining,foreachprinciple,thescientificdata
whichmakeitpossibletoidentifyandcharacterizethe
productsconcernedandthestudiesnecessaryinorderto
evaluatetheirnutritionalpropertiesandbiological
effects;whereastheseguidelinesmustbeapplicableon
thedateonwhichDirective82/471/EECitselfenters
intoforce;
Whereastheguidelinesareintendedprimarilyasageneral
guide;whereas,dependingonthenatureoftheproductor
itsconditionsofuse,theextentofthestudiesnecessaryin
ordertoevaluateitspropertiesoritseffectsmayvary;Article3
ThisDirectiveisaddressedtotheMemberStates.
DoneatLuxembourg ,18April1983.
FortheCouncil
ThePresident
I.KIECHLE
(')OJNoL213,21.7.1982,p.8.

NoL126/24 OfficialJournaloftheEuropeanCommunities 13.5.83
ANNEX
GUIDELINES FORTHEASSESSMENT OFCERTAIN PRODUCTS USEDINANIMAL
NUTRITION
Generalaspects
These'guidelines'constituteaguideintendedtoestablishdossiersonproductslistedinitems1.1and1.2of
theAnnextoDirective82/471/EEC,whichhavebeenobtainedfromculturingmicro-organisms and
whicharelikelytobeadmittedasanewsourceofproteinsinanimalnutrition.Thesedossiersshouldenable
anassessmentofsuchproductsbasedonthepresentstateofknowledgeandshouldensuretheircompliance
withthefundamentalprincipleslaiddownforpermittingtheiruse,whicharethesubjectofArticle6(2)of
theabovementioned Directive.
Allthestudiesoutlinedinthisdocumentmayberequiredand,ifnecessary,additionalinformationmaybe
requested.Asageneralrule,alltheinformationnecessarytoestablishtheidentityofthemicro-organism
andthecompositionoftheculturemedium,andalsothemanufacturing process,characteristics ,
presentation ,conditionsofuse,methodsofdeterminationandnutritionalpropertiesoftheproductmust
beprovided.Thesameappliestotheinformationnecessarytoassessthetoleranceoftheproductbythe
targetspeciesandtherisksformanandtheenvironment ,whichcouldresultdirectlyorindirectlyfromthe
useoftheproduct.Thetoxicologicalstudiesrequiredforthispurposewilldependonthenatureofthe
product,theanimalspeciesconcernedandthemetabolismoftheproductinlaboratoryanimals.
Thedocumentation tobeprovidedshouldincludedetailedreports,presentedintheorderandwiththe
numberingproposedintheseguidelinesandshouldbeaccompaniedbyasummary.Theomissionofany
proposedstudiesshouldbejustified.Thepublicationsquotedasreferencesshouldbeattached.
Observations
Theterm'product',asusedintheseguidelines,referstoanyproteinaceousproductinthestateinwhichit
willbepresentedasfeedingstufforcomponentofafeedingstuff .
Anymodificationinthemanufacturing processorintheconditionsofuseofaproductwillrequire
notificationand,ifnecessary,additionaldocumentation foranewassessment .
Presentation ofstudies
I.Micro-organism ,culturemediumandmanufacturing process,characteristicsofproduct,presentation
andconditionsofuse,methodsofdetermination
II.Studiesonthenutritionalpropertiesoftheproduct
III.Studiesonthebiologicalconsequencesoftheuseoftheproductinanimalnutrition
IV.Otherrelevantstudies
SECTION I
MICRO-ORGANISM CULTUREMEDIUMANDMANUFACTURING PROCESS,
CHARACTERISTICS OFPRODUCT,PRESENTATION ANDCONDITIONS OFUSE,METHODS
OFDETERMINATION
1.MICRO-ORGANISM
1.1.Classification ,provenance,morphology ,biologicalproperties,anygeneticmanipulation .
1.2.Innocuity,possiblesurvivaloutsidethefermenterandanyenvironmentalconsequences .
1.3.Constancyandpurityofstrainscultivated.Methodsusedtocheckthesecriteria.
2.CULTUREMEDIUMANDMANUFACTURING PROCESS
2.1.Compositionofsubstrate,addedsubstances,,etc.
2.2.Manufacturing ,dessicationandpurificationprocesses.Devitalizingprocessformicro-organisms .
Methodsusedtochecktheconstancyofcompositionofthecultureproductandthedetectionofany
chemical,physicalandbiologicalcontamination duringproduction .
2.3.Technicalprocessesofpreparationforuse.

13.5.83 OfficialJournaloftheEuropeanCommunities NoL126/25
3.CHARACTERISTICS OFPRODUCT
3.1.Physicalandphysico-chemical properties :macro-andmicro-morphology ,particlesize,density,
specificweight,hygroscopicity ,solubility,electrostaticproperties,etc.
3.2.Chemicalcompositionandcharacteristics .
3.2.1.Contentofmoisture,crudeprotein,crudefat,crudecellulose,crudeash,carbohydrates .Limitsof
variationofthesecontents.
3.2.2.Contentoftotalammonium ,amide,nitrateandnitritenitrogen,nucleicacids,protein.Qualitative
andquantitativecompositionoftotalandfreeaminoacids,andpurineandpyrimidinebases.
3.2.3.Qualitativeandquantitativecompositionoftotallipids:fattyacids,non-saponifiable matter,lipid
solublepigments,phospholipids .
3.2.4.Compositionofthecarbohydratefraction.
3.2.5.Qualitativeandquantitativecompositionofinorganiccomponents .
3.2.6.Qualitativeandquantitativecompositionofvitamins.
3.2.7.Qualitativeandquantitativecompositionoftheotherconstituents :additives,residuesofsubstrate
andsolvents,otherpotentiallyharmfulresiduesofthemetabolismofthesubstrate,oftheculture
medium,ofthemanufacturing process.
3.3.Microbiological contamination oftheproduct.
3.4.Behaviourandstabilityoftheproduct,assuchandwhenmixedwithfeedingstuffsincurrentuse,
duringstorage.
4.PRESENTATION ANDCONDITIONS OFUSE
4.1.Proposednamesofmarketingtheproduct.
4.2.Proposedformulationsformarketingtheproduct.
4.3.Intendeduseoftheproductinanimalnutrition.Intendedconcentrations inthecomplete
feedingstuffsandintheintendedquantitiesinthedailyrationsfortheanimalspecies
concerned .
5.METHODSOFDETERMINATION
Qualitativeandquantitativemethodsfordetermination oftheproductincompleteand
complementary feedingstuffs .
NB:Descriptionofthesemethodsshouldbeaccompaniedbyinformationastospecificity,
sensitivity,limitsofdetection,marginoferror,possibleinterferencesbyothersubstances.
Samplesoftheproductinitsvariousproposedpresentationsshouldbeavailable.
SECTIONII
STUDIESONTHENUTRITIONAL PROPERTIES OFTHEPRODUCT
1.ASSESSMENT OFPROTEINVALUE
1.1.Chemical,biochemicalandmicrobiological studies.
1.2.Studiesonlaboratoryanimals,comparedwithreferenceproteins.
2.STUDIESONTARGETSPECIES
Thefollowingstudiesshouldbeperformedoneachtargetspeciesincomparisonwithacontrol
groupreceiving,underthesameconditionsofnutritionalbalance,adietincurrentusecontaining
equivalentamountsofproteinnitrogen,forruminantsoftotalnitrogen.
2.1.Proteinandenergysupplementation valueoftheproductintherationsundertheproposed
conditionsofuseatvariousphysiologicalstagesoftheanimals(e.g.growingperiod,pregnancy,
laying).
2.2.Influenceoftheproductundertheproposedconditionsofuseongrowthrate,feedconversionrate,
morbidity,mortality.
2.3.Optimumnutritionallevelsofincorporationoftheproductintherations.
2.4.Effectoftheproductundertheproposedconditionsofuseonthetechnological ,organolepticor
otherqualitiesofedibleproductsofanimalorigin.
3.EXPERIMENTAL CONDITIONS INTHESTUDIESONTARGETSPECIES
Giveadetaileddescriptionofthetestsperformedandprovidethefollowingdata:
3.1.Species,breed,ageandsexoftheanimals,identificationprocedure.

NoL126/26 OfficialJournaloftheEuropeanCommunities 13.5.83
3.2.Numberoftestandcontrolgroups;numberofanimalsineachgroup(thenumbershouldbelarge
enoughforstatisticalanalysisusingsuitablestatisticalparameters).
3.3.Levelsofincorporationoftheproduct,qualitativeandquantitativecompositionoftherationand
itsanalysis.
3.4.Locationofeachexperiment ,physiologicalstateandanimalhealthconditions,rearingconditions
(theseshouldreflectthoseusedinpracticeintheCommunity).
3.5.Exactdurationoftestinganddateoftheanalysesperformed.
3.6.Adverseeffectswhichoccurredduringtheexperimentandtimeoftheirappearance .
SECTIONIII
STUDIESCONCERNING THEBIOLOGICAL CONSEQUENCES OFTHEUSEOFTHEPRODUCT
INANIMALNUTRITION
Thestudiesoutlinedinthissectionareintendedtopermitassessmentofthesafetyinuseoftheproductin
thetargetspecies,andoftherisksformanandtheenvironmentwhichcouldresultdirectlyorindirectly
fromthisuse.Thetoxicologicalstudiesrequiredforthispurposewilldependonthenatureoftheproduct,
theanimalspeciesconcernedandthemetabolismoftheproductinlaboratoryanimals.
1.STUDIESONTARGETSPECIES
Thefollowingstudiesshouldbeperformedoneachtargetspeciesincomparisonwithacontrol
groupreceiving,underthesameconditionsofnutritionalbalance,adietincurrentusecontaining
equivalentamountsofproteinnitrogen,forruminantsoftotalnitrogen.
1.1.Maximumincorporationratesoftheproductinthedailyrationwithoutproducinganyadverse
effect.
1.2.Possibleeffectoftheproductonfertilityandreproduction ,ifappropriate .
1.3.Effectsofingestionoftheproductundertheproposedconditionsofuseonmicro-organisms ofthe
floraofthealimentarytractandoncolonizationofpathogensinthealimentarytract.
1.4.Investigationundertheproposedconditionsofuseofpossibleresiduesoftheproduct(substrate,
culturemedium,solvents,contaminants )inedibleproductsofanimalorigin.
1.5.Investigationundertheproposedconditionsofuseofpossibleresiduesoftheproduct(substrate,
culturemedium,solvents,contaminants )inexcreta.
2.STUDIESONLABORATORY ANIMALS
2.1.Metabolism
Fateoftheproductintheanimal:absorption,accumulation ,biotransformation ,elimination .
2.2.Mutagenicity
Investigations ofpotentialmutagenicityduetocontaminants (inparticularmycotoxinsand
bacteria)orresiduesoftheproduct(substrate,culturemedium,solvents)includinginvitro
screeningtestsusingmetabolicactivationsystems.
2.3.Toxicological studies
Thefollowingstudiesshouldbeperformedincomparisonwithcontrolgroupsreceiving,underthe
sameconditionsofnutritionalbalance,adietincurrentusecontainingequivalentamountsof
proteinnitrogen.Toxiceffectsshouldbeinvestigatedtoelucidatetheircauseandmechanismsand
toascertainthattheydonotresultfromnutritionalimbalanceorfromanoverdosageoftheproduct
inthediet.
2.3.1.Subchronictoxicity(atleast90days)
Ingeneral,thesestudiesshouldbecarriedoutontwoanimalspecies,oneofwhichbeingarodent.
Theproductshouldbeadministeredinthedailyrationinatleasttwolevelsofincorporation .These
shouldbechosensoastodetermine,ifpossible,ano-effectlevelandalevelshowingsomeadverse
effect.Theanimalgroupsshouldcontainanadequatenumberofsubjectsofeachsex.Acontrol
groupshouldalwaysbeincluded.
Allrelevantbiologicaldatashouldberecordedatappropriateintervals,particularlydataongrowth
rate,feedconsumption ,haematology ,urineanalysis,biochemicalparameters,mortality,organ

13.5.83 OfficialJournaloftheEuropeanCommunities NoL126/27
weights,grosspathologyandhistopathology ofmajororgansandtissues.Theresultsshouldbe
presentedindetailand,asfaraspossible,shouldincludestatisticalassessment .
2.3.2.Chronictoxicity
Ingeneral,chronictoxicitystudiesshouldbecarriedoutontwoanimalspecies,oneofwhichbeing
arodent.Theproductshouldbeadmininstered inthedailyrationinatleasttwolevelsof
incorporation .Experimentsshouldextendforaminimumoftwoyearsintherator80weeksin
mice.Theanimalgroupsshouldcontainanadequatenumberofsubjectsofeachsex.Acontrol
groupshouldalwaysbeincluded.
Thebiologicalexaminationsmentionedunderpoint2.3.1shouldbecarriedoutpreferablyona
smallsatellitegroupofanimals(agroupseparatedfromanddependentuponthemaingroup)at
appropriateintervalsthroughouttheexperimentandonthesurvivinganimalsattheendofthe
experiment .
2.3.3.Carcinogenicity
Forassessingcarcinogenicity ,particularattentionshouldbepaidtothetimeofappearance ,the
histologicaltypesofanyobservedtumoursandtheirincidence.Anyeffectontheincidenceof
tumoursand/ortheincidenceorprogressofdiseasesshouldbeassessedbyreferencetocontrol
groups,asindicatedinparagraph2.3.Theresultsshouldbepresentedindetailand,asfaras
possible,shouldincludestatisticalassessment .
2.4.Otherstudies
Reproduction studiesshouldextendoveratleasttwofilialgenerationsandmaybecombinedwith
embryotoxicity includingteratogenicity studies.Particularattentionshouldbepaidtofertility,
fecundityandobservationonpost-nataldevelopmentoflitters.Anyothermethodthatis
scientificallyjustifiableandlikelytoproducemeasurableresults(e.g.relaytoxicity)maybe
provided.
2.5.Experimentalconditionsinthestudiesonlaboratoryanimals
Givedetaileddescriptionofthetestsperformedandprovidethefollowingdata:
2.5.1.Species,breed,strainandsexofanimals.
2.5.2.Numberoftestandcontrolgroups,numberofanimalsineachgroup(thenumbershouldbelarge
enoughforstatisticalanalysisusingsuitablestatisticalparameters).
2.5.3.Levelsofincorporationoftheproduct,qualitativeandquantitativecompositionoftherationand
itsanalysis.
2.5.4.Generalrearingconditionsthroughouttheperiodoftesting.
2.5.5.Exactdurationoftestinganddateofexaminationsperformed .
2.5.6.Rateandtimingofdeathsforthevarioustestgroups.
2.5.7.Clinicalsymptomsandpathologicalalterationswhichoccurredduringtheexperimentandtimeof
theirappearance .
3.STUDIESCONCERNING THEENVIRONMENT
Dependingonthenatureofpossibleresiduesoftheproduct(substrate,culturemedium,solvents,
contaminants )inexcretaoftargetspecies,dataonthefateoftheseresiduesinmanure,soiland
waterandalsotheireffectsonsoilbiology,plantgrowthandaquaticlifemayberequired.
SECTIONIV
OTHERRELEVANT STUDIES
Dependingonthenatureandtheconditionsofuseoftheproduct,dataonallergiceffects,onirritationof
theskinandmucusmembranesoftheeye,respiratoryordigestivetractmayberequiredtoassesspossible
risksinhandlingtheproductandtopreventthem.