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1、55.2.2ToXiCOIOgiCalASSeSSment毒理学评价55.2.3ApplicationofaThresholdofToxicologicalCOnCern毒理学担忧阈值应用55.3DecisionTreefor.Assessmentof.AcceptabilityofGenotoxicImpurities基因毒性杂质可接受性评价决策树7REFERENCES.参考文献8EXECUTIVESUMMARY内容摘要Thetoxicologicalassessmentofgenotoxicimpuritiesandthedeterminationofacceptable1imitsfor
2、suchimpuritiesinactivesubstancesisadifficultissueandnotaddressedinsufficientdetai1intheexistingICHQ3Xguidances.ThedatasetusualIyavailableforgenotoxicimpuritiesisquitevariableandisthemain物中可接受的杂质水平。较高的临界值可以在特定的条件下，如短期暴露周期等，进行推算。1. INTRODUCTION介绍generalconceptofqualificationofimpuritiesisdescribedinth
3、eguidelinesforactivesubstances(Q3A,ImpuritiesinNewActiveSubstances)ormedicinalproducts(Q3B,ImpuritiesinNewMedicinalProducts),wherebyqualificationisdefineclastheprocessofacquiringandevaluatingdatathatestablishesthebiologicalsafetyofanindividualimpurityoragivenimpurityprothelevel(三)specified.Inthecase
4、ofimpuritieswithagenotoxicpotential,determinationofacceptabledoselevelsisgeneralIyconsideredasaparticularlycriticalissue,whichisnotspecificallycoveredbytheexistingguideines.在原料药(Q3A)和药物制剂(Q3B)的杂质指导原则中，杂质限度确定的依据包括各个杂质的生物平安性数据或杂质在某特定含量水平的探讨概况。而对于遗传毒性杂质限度的确定，通常都认为是特殊关键的问题，但目前尚无相关的指导原则。2. SCOPE范围ThisGui
5、delinedescribesageneralframeworkandpracticalapproachesonhowtodealwithgenotoxicimpuritiesinnewactivesubstances.Italsorelatestonewapplicationsforexistingactive目前对于基因毒性杂质的分类主要是指：在以DNA反应物质为主要探讨对象的体内体外试验中，假如发觉它们对DNA有潜在的破坏性，那可称之为基因毒性。假如有足够的后续试验，可由单独的体外试验结果，对它的体内关联性进行评估。在缺乏这样的信息时，体外基因毒性物质常常被考虑为假定的体内诱变剂和致癌剂
6、。3. 1.EGA1.BASIS法规依据ThisguidelinehastobereadinconjunctionwithDirective2019/83/EC(asamended)andallrelevantCHMPGuidancedocumentswithspecialemphasison:在阅读该指南时有必要参考Directive201983ECm以及相关的CHMP指南文件，特殊是以下几个指南：ImpuritiesTestingGuideline:ImpuritiesinNewDrugSubstances(CF,MPICH273799,ICHQ3(R)NoteforGuidanceonI
7、mpuritiesinNewDrugProducts(CPMP/ICH/2738/99,ICHQ3B(R)NoteforGuidanceonImpurities:ResidualSolvents(CPMP/ICll/283/95)NoteforGuidanceonGenotoxicity:GuidanceonSpecificAspectsofRegulatoryGenotoxicityTestsforPharmaceuticals(CPMP/ICH/141/95,ICHS2A)然而，对于一些遗传毒性事务，其产生生物学意义的阈值效应的机理正越来越为人所了解。对于非DNA靶点的化合物和潜在致突变剂
8、更是如此,因为它们在及关键靶点接触前就已经去毒化了。对于这些化合物，探讨的基础可以是确定关键的未视察到影响的剂量（NOE1.）和采纳不确定因子。EvenforcompoundswhichareabletoreactwiththeDNAmolecule,extrapolationinalinearmannerfromeffectsinhigh-dosestudiestoverylowlevel（human）exposuremaynotbejustifiedduetoseveralprotectivemechanismsoperatingeffectivelyatlowdoses.However,
9、atpresentitisextremelydifficulttoexperimentallyprovetheexistenceofthresholdforthegenotoxicityofagivenmutagen.Thus,intheabsenceofappropriateevidencesupportingtheexistenceofathresholdforagenotoxiccompoundmakingitdifficulttodefineasafedoseitisnecessarytoadoptaconceptofalevelofexposurethatcarriesanaccep
10、tablerisk.即使对能及DNA分子发生反应的化合物，由于低剂量时有多种有效的爱护机制存在，而不能将高剂量卜的影响以线性方式外推到很低的（人）暴露水平。不过，目前要用试验方法证明某诱变剂的遗传毒性阈值仍旧特别困难。所以，在缺乏恰当的证据支持遗传毒性阈值存在的状况卜.，确定平安剂量很困难，因此特别有必要采纳一个可接受风险的暴露水平概念。5.RECOMMENDATIONS建议ASstatedintheQ3guideline,actualandpotentialimpurities指导原则认为这些探讨采纳含有那些需限制杂质的原料药进行是可行的，但用分别出来的杂质进行这些探讨更恰当，也是高度举荐的
11、方法。FordeterminationofacceptablelevelsofexposuretogenotoxiccarcinogensConsiderationsofpossiblemechanismsofactionandofthedose-responserelationshipareimportantcomponents.Basedontheaboveconsiderationsgenotoxicimpuritiesmaybedistinguishedintothefollowingtwoclasses:依据以上论述，遗传毒性杂质可以归纳成以卜两类：-Genotoxiccompoun
12、dswithsufficient(experimental)evidenceforathreshold-relatedmechanism有充分阈值相关机理证据(试验)的遗传毒性化合物-Genotoxiccompoundswithoutsufficient(experimental)evidenceforathreshold-relatedmechanism无充分阈值相关机理证据(试验)的遗传毒性化合物5.1 GenotoxicCompoundsWithSufficientEvidenceforaThreshold-RelatedMechanism具有充分证据证明其阈值相关机理的基因毒性化合物E
13、xamplesofmechanismsofgenotoxicitythatmaybedemonstratedtoleadtonon-linearorthresholdeddose-responserelationshipsincludeinteractionwiththespindleapparatusofcelldivisiondifferentstartingmaterials.Thismightforinstanceincludecaseswherethestructure,whichisresponsibleforthegenotoxicand/orcarcinogenicpotent
14、ialisequivalenttothatneededinchemicalsynthesis(e.g.alkylationreactions).须要供应充分的论证来说明没有可行的替代方法存在，包括可替代的合成路途或配方，不同的起始物料等。比如，应证明具有遗传毒性和/或致癌性的结构在化学合成中(如烷化反应)是必需的。Ifagenotoxicimpurityisconsideredtobeunavoidableinadrugsubstance,technicalefforts(e.g.purificationsteps)shouldbeundertakentoreducethecontentoft
15、hegenotoxicresiduesinthefinalproductincompliancewithsafetyneedsortoalevelaslowasreasonablypracticable(seesafetyassessment).DataonchemicalstabiIityofreactiveintermediates,reactants,andothercomponentsshouldbeincludedinthisassessment.假如遗传毒性杂质在原料中不行避开，则应当实行适当的技术(如纯化步骤)降低该杂质的含量，以满意平安性要求，或符合“合理可行的最低限量”原则(
16、见平安评估)。药学评估还应包括反应中间体、反应物和其它组件等的化学稳定性探讨。Detectionand/orquantificationoftheseresiduesshouldbedonebystate-of-the-artanalyticaltechniques.应当运用比较先进的分析检测技术来检测和量化这些残留的杂质。Inmostcasesoftoxicologicalassessmentofgenotoxicimpuritiesonlylimiteddatafrominvitrostudieswiththeimpurity(e.g.Amestest,chromosomalaberrat
17、iontest)areavailableandthusestablishedapproachestodetermineacceptableintakelevelscannotbeapplied.Calculationofasafetymultiplesfrominvitrodata(e.g.Amestest)areconsideredinappropriateforjustificationofacceptable1imits.Moreover,negativecarcinogenicityandgenotoxicitydatawiththedrugsubstancecontainingthe
18、impurityatlowppmlevelsdonotprovidesufficientassuranceforsettingacceptable1imitsfortheimpurityduetothelackofsensitivityofthistestingapproach.Evenpotentmutagensandcarcinogensaremostlikelytoremainundetectedwhentestedaspartofthedrugsubstance,i.e.atverylowexposurelevels.Apragmaticapproachisthereforeneede
19、dwhichrecognisesthatthepresenceofverylowlevelsofgenotoxicimpuritiesisnotassociatedwithanunacceptablerisk.大多数状况下，遗传毒性杂质的毒理学评估只是局限于杂质的体外探讨(如AineS试验，染色体畸变试验)，但这些方法并不适用于确定杂质可接受的摄入水平。也就是说，依据体外数据(如AmeS试验)计克杂质的“平安倍数(SafetymUltiPIeS)”、进而确定可接受的限度，是不合适的。此外，用含有较低(PPm级)杂质水平的原料药探讨其致癌性和遗传毒性，即使得出阴性结果也不足以确保该杂质限度的合理
20、性，因为这种试验方法缺少必要的灵敏度。有些具有很强致突变性和致癌性物质及原料药一起进行试验时，因为在特别低的暴露水平状况下，很有可能因为低于检测限而无法检出。所以，假如相识到含量特别低的遗传毒性杂质不存在“不行接受的风险”(UnaCCePtabIerisk),那么可以实行好用的方法来限制该杂质。5.2 .3ApplicationofaThresholdofToxicologicalConcern毒理学相关的阈值应用Athresholdoftoxicologicalconcern(TTC)hasbeendevelopedtodefineacommonexposurelevelforanyunst
21、udiedchemicalthatwi11notposeariskofsignificantcarcinogenicityorothertoxiceffects(Munroetal.2019,KroesandKozianowski2019).ThisTTCvaluewasestimatedtobe1.5ug/PerSOn/day.TheTTC,originallydevelopedasauthresholdofregulationnattheEDAforfoodcontactmaterials(Rulis1989,FDA1995)wasestablishedbasedontheanalysis
22、of343carcinogensfromacarcinogenicpotencydatabase(Goldetal.1984)andwasrepeatedlyconfirmedbyevaluationsexpandingthedatabasetomorethan700carcinogens(Munro1990,Cheesemanetal.2019,Kroesetal.2019).Theprobabilitydistributionofcarcinogenicpotencieshasbeenusedtoderiveanestimateofadailyexposurelevel(ug/person
23、)ofmostcarcinogenswhichwouldgiverisetolessthanaoneinami11ion(1x106)upperboundlifetimeriskofcancer(*virtualIysafedose).Furtheranalysisofsubsetsofhighpotencycarcinogensledtothesuggestionofa10-foldlowerTTC(0.15gday)forchemicalswithstructuralalertsthatraiseconcernforpotentialgenotoxicity(Kroesetal.2019)
24、.“毒理学关注的阈值”用于定义那些不会产生显著致癌性或其他毒性作用、但乂未明确探讨的化合物的“常见暴露量”(commonexposurelevel)(Munroetal.2019,KroesandKozianowski2019)0该TTC估计值是1.5ug人/日。TTC概念最早来源于FDA关于食品接触材料的“规定阈值”(athresholdofregulation)(Rulis1989,FDA1995),该阈值依据对致癌实力数据库(Goldetal.1984)中343种致癌物质的分析结果得出。随后该数据库扩大到700多个致癌性物质(MUnrO1990,Cheesemanetal.2019,Kr
25、oesetal.2019),这种分析结果不断得到重复验证。通过对致癌实力的概率分布进行评价，可以得到一个对大多数致癌物质适用的“日常摄入水平(Ug/person)”，此水平造成的一生中患癌症的风险小于正常风险水平的上限1X10-6(真实的平安剂量)。对于含有“可能引起遗传毒性结构”的化合物，其TTC应严格10倍(0.15Hg/日)(Kroesetal.2019)oHowever,forapplicationofaTTCintheassessmentofacceptableIimitsofgenotoxicimpuritiesindrugsubstancesavalueof1.5gday,cor
26、respondingtoa10-5lifetimeriskofcancercanbejustifiedasforpharmaceuticalsabenefitexists.Itshou1dberecognizedinthiscontextthatthemethodsonwhichtheTTCvalueisbased,aregenerallyconsideredveryconservativesincetheyinvolvedasimple1inearextrapolationfromthedosegivinga50%tumourincidence(TD50)toa1in106incidence
27、,usingTD50dataforthemostsensitivespeciesandmostsensitivesite(several,4worstcaseassumptions)(Munroetal.2019).然而，用11C评估原料药中的遗传毒性杂质限度，1.5g日(相当于10万分之一的患癌风险)是可以接受的。应当承认，基于TTC值限制遗传毒性杂质是特别保守的，因为这只是依据从产生50%肿瘤发生率(TD50)到百万分之一样癌率的剂量线性推导得到的，而且TD50数据是用最敏感的动物和最敏感的部位探讨得到的(儿个“最坏条件”假设)(Munroeta1.2019)oSomestructural
28、groupswereidentifiedtobeofsuchhighpotencythatintakesevenbelowtheTTCwouldbeassociatedwithahighprobabiIityofasignificantcarcinogenicrisk(Cheesemanetal.2019,Kroesetal.2019).Thisgroupofhighpotencygenotoxiccarcinogenscomprisesaflatoxin-like-,nitroso-,andazoxy-compoundsthathavetobeexcludedfromtheTTCapproa
29、ch.Riskassessmentofmembersofsuchgroupsrequirescompound-specifictoxicitydata.有几个结构基团被认定为具有特别高的基因毒性，它们即使被摄入低于TTC值的量也会面临特别高的基因毒性风险（CheeSemanetal.2019,Kroesetal.2019)o这些高致癌性物质包括黄曲霉素类、N-亚硝基物和偶氮类化合物，不适用TTC方法。这类化合物的风险评估需采纳特地的毒性数据。TheremaybereasonstodeviatefromtheTTCva1uebasedontheprogenotoxicityresults.依据基
30、因杂质概况，有些状况卜会偏离TTC值。PositiveresultfrominvitrostudiesonlymayallowtoexemptanimpurityfromlimitationatTTCleveliflackofinvivorelevanceofthefindingsisconvincinglydemonstratedbasedonaWeight-Ofevidenceapproach(seeICHS2guidelines).Thisapproachwillusuallyneednegativeresultswiththeimpurityfromsomeadditionalinvi
31、troand/orappropriateinvivotesting.假如依据证据权衡法能充分证明“结果缺乏体内相关性”，体外试验的阳性结果也仅能在TTC水平上解除一个杂质(参见ICH指南S2)。这种方法常常须要在额外的体外试验和/或合理的体内试验，并且得到杂质的阴性结果。TTCvaluehigherthan1.5g/daymaybeacceptableundercertainconditions,e.g.short-termexposure,fortreatmentofalife-threateningcondition,whenlifeexpectancyislessthan5years,o
32、rwheretheimpurityisaknownsubstanceandhumanexposurewi11bemuchgreaterfromothersources(e.g.food).GenotoxicimpuritiesthatarealsosignificantmetabolitesmaybeassessedbasedontheacceptabiIityofthemetabolites.某些状况下11c值高于1.5Ug/日也是可以接受的，如短期用药；用于治疗威逼生命疾病的药物；或人的存活期少于5年：或该杂质是已知物质，人体从其他途经(如食物)获得的暴露量远远高于药物途经。假如遗传毒性杂
33、质本身就是重要的代谢物，那么该杂质可以依据代谢物的可接受限度进行限制。Theconcentrationlimitsinppmofgenotoxicimpurityindrugsubstancederivedfromthe11Ccanbecalculatedbasedontheexpecteddailydosetothepatientusingequation(1).采纳下列公式，从TTe值和日服用剂量，可以计算出原料药中的基因毒性杂质的浓度限度。(1)Concentrationlimit(ppm)=TTCgdaydose(gday浓度限度(PPm)=TTCUgday/剂量(gdayTheTTC
34、conceptshouldnotbeappliedtocarcinogenswhereadequatetoxicitydata(long-termstudies)areavaiIableandallowforacompound-specificriskassessment.对于有准确毒性数据(长期毒性探讨)的致癌性物质不宜运用TTC概念，应进行特定化合物风险评估。IthastobeemphasizedthattheTTCisapragmaticriskmanagementtoolusingaprobabiIisticmethodology,i.e.thereisahighprobabiIity
35、thata10-5lifetimecancerriskwi11notbeexceededifthedailyintakeofagenotoxicimpuritywithunknowncarcinogenicpotential/potencyisbelowtheTTCvalue.TheTTCconceptshouldnotbeinterpretedasprovidingabsolutecertaintyofnorisk.应强调，TTC是一个好用性的风险管理方法，是按概率方法学估算的。比如按这概念，假如某未知致癌性遗传毒性杂质的摄入量低于TTC值，那么就可以保证患癌风险限制在I万分之一之内。但TT
36、C概念不能被理解为确保肯定无风险。5. 3DecisionTreeforAssessmentofAcceptabilityofGenotoxicImpurities基因毒性可接受性评价决策树(shadedboxes=pharmaceuticalassessment,whiteboxes=toxicologicalassessment)(阴影框=药学评价，白框=毒理学评价)1) Impuritieswithstructuralrelationshiptohighpotencycarcinogens(seetext)aretobeexcludedfromtheTTCapproach1)结构上及高致
37、癌性物质有关的杂质(见正文)不能采纳TTC法。2) Ifcarcinogenicitydataavai!able:Doesintakeexceedcalculated10-5cancerlifetimerisk?2)假如有致癌性数据：摄入量超过10-5患癌风险吗？3) Case-by-caseassessmentshouldincludedurationoftreatment,indication,patientpopulationetc(seetext)3)具体状况具体分析，包括用药时间长短、适应症、患者人群等(见正文)。*)Abbreviations:缩写N0E1./UF-NoObserv
38、edEffect1.eve!/UncertaintyFactor,未视察到效果水平/不确定因素PDE-PermittedDailyExposure,允许日接触量TTC-ThresholdofToxicologicalConcern毒理学关注的域值REFERENCES参考文献CheesemanM.A.,MachugaE.J.,BaileyA.B.,Atieredapproachtothresholdofregulation,EoodChemToxicol37,387412,2019食品化学毒性，2019,DoboK.1.,GreeneN.,CyrM.0.,CaronS.,KuW.W.,Theap
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