基因毒性小册子

遗传毒性杂质遗传毒性：泛指各种因素〔物理、化学因素〕与细胞或生物体的遗传物质发生作用而产生的毒性.1、致突变性：与DNA相互作用产生直接潜在的影响,使基因突变〔bacteria reverse mutation 〔Ames〕试验〕2、致癌性：具有致癌可能或倾向〔需要长期研究！〕3、警示结构特征：一些特殊的结构单元具有与遗传物质发生化学反响的能力,会诱导基因突变或者导致染色体重排或断裂,具有潜在的致癌风险.遗传毒性物质：在很低的浓度下即可诱导基因突变以及染色体的断裂和重排, 因此具有潜在的致癌性.EMA通告〔1〕、具体事项：1、哪些品种中会出现甲磺酸酯〔或甲磺酸烷基酯〕.特别是甲磺酸盐等形式的API或其合成中用到甲磺酸的API,甲磺酸烷基酯-甲磺酸甲酯、乙酯、其它低级醇酯,应认定为潜在杂质.2、羟乙基磺酸盐、苯磺酸盐、对甲苯磺酸盐的API.应说明类似物质磺酸烷基酯或芳基酯污染的危险.3、限度要求：无其它毒性数据时,这些高风险杂质应依据TTC设定限度.1.5小我g为单位的最大日剂量得ppm限度.4、法律依据：EP专论要求凡以甲磺酸盐和羟乙基磺酸盐形式存在的API,均应在其生产过程中采取以下平安举措：必须对生产工艺进行评估以确定家磺酸烷基酯〔羟乙基磺酸烷基酯〕形成的可能,特别是反响溶媒含低级醇的时候,很可能会出现这些杂质.必需时需对生产工艺进行验证以说明在成品中未检出这类杂质.〔2〕、落实举措：1、API生产是否涉及在甲磺酸〔羟乙基磺酸盐、苯磺酸盐、对甲苯磺酸等低分子量磺酸〕或相应酰氯存在下,使用甲醇、乙醇、正丙醇、异丙醇等低级脂肪醇〔如甲醇、乙醇、正丙醇、异丙醇等〕.2、对相应酯形成的可能性是否降到最低.3、是否有有效的去除精制步骤.设备清洗-是否设计的低级脂肪醇的使用〔方法,TTC限度〕？起始物料〔低分子量磺酸盐或酰氯〕中是否限制了其低级脂肪醇酯〔方法,TTC限度〕？当被磺酸酯或相关物质污染的磺酸用于API合成时能否保证其中潜在的遗传毒性杂质不超过TTC?应考虑各种烷基或芳基磺酸酯杂质累积的风险.API最后一步用到的磺酸衍生物,应进行风险分析.收回溶剂：中的磺酸酯类杂质〔如乙醇中的EMS、甲醇中的MMS、异丙醇中的IMS〕的富集和残留是否进行了限制.制剂过程：甲磺酸盐等相关形式的API的制剂过程〔制粒、包衣…〕是否使用了醇,是否有足够灵敏的方法监控只集中杂质处于TTC水平！储存过程：甲磺酸盐等相关形式的API及制剂储存过程中是否会存在形成烷基或芳基磺酸酯.整改：存在风险必须研究,报告监管部门并按相关程序提交变更申请.遗传毒性杂质研究相关指导原那么ICH Q3A 〔R2〕、ICH Q3B 〔R2〕〔遗传毒性杂质仅有提及〕EMA : Guideline on the Limits of Genotoxic Impurities, 2006.EMA : Questions and Answers on the CHMP Guideline on the limits of genotoxic Impurities, 2021.FDA : Guidance for Industry:Genotoxic and Carcinogenic Impurities in Drug Substances and Products:Recommended Approaches. Draf2021.ICH M7 : Draft consensus guideline assessment and control of DNA reactive〔Mutagenic〕impurities in pharmaceutical to limit potential carcinogenic riskICH M7目前状态：2021年2月6日发布,征求意见历时17个月〔一般6个月〕后,于2021年7月15日正式发布.现在进入ICH区域实施期〔在美国联邦注册公布,经CHMP和欧盟委员会批准,在日本译〕指南比拟复杂,需要企业和卫生部门调整程序,因此,预期在ICH公布后18个月前方可全面实施M7.与指南一起发布了实施指导：①M7发布后的AMES试验需立即实施M7标准.②M7发布前乙月青开始2b/3期试验的工程可以继续,结构活性关系定量分析、杂质评估和文件记录不是必须的.③M7发布后的上市申请,如未进行2b/3期试验,须在36个月内完成上述试验.④……ICH M7适用范围：①主要用于新药〔原料药、制剂J〕的临床研究阶段及上市注册申请.包括孤儿药,新复方制剂,新制剂,新剂型的申请. 〔原料药没有改变,不做回忆性评价〕②也适用于上市后的相关变更,包括临床使用的变化.A.上市后API生产变更：合成路线、试剂、溶剂以及工艺条件的变更等可能导致新的遗传毒性杂质产生或已有的遗传毒性杂质含量发生改变.B.上市后制剂的变更：处方、生产工艺、剂型、规格改变可能导致新的遗传毒性杂质或已有的遗传毒性杂质含量发生改变.C.上市后药品的临床使用发型变更：剂量的显著增加、使用时间的增加、适应症的变更〔例如,从危及生命的适应症变为一般严重适应症〕,要求对遗传毒性杂质的限度进行重新评估.在剂量或者治疗时间不改变的情况下,已上市药品增加新的给药途径或者扩大患者人群〔即使包括孕妇和/或儿童〕在临床使用中的变更通常不要求重新评估.③依照ICH S9指导原那么的抗月中瘤新药的申请不适用M70〔以健康人进行药代研究除外〕ICH M7要答复的问题：①提供了一个可用于遗传毒性杂质鉴别、分类、定量分析和限制的可行性框②限制潜在的致癌风险③提供平安性评价和质量风险限制的理念④对ICHQ3A和Q3B的补充I：如何根据结构活性分析评估杂质潜在的遗传毒性？II.如何确定杂质TTC?遗传毒性杂质的可接受水平是多少？III.为什么潜在基因毒性物质具有相似的分子结构？IV.是否可以采用毒理学关注阈值〔TTC〕来规定遗传毒性杂质的水平？V.计算TTC时,是否可以将多个遗传毒性杂质合并计算？VI.哪些是可接受的特殊情况？日摄入量大于TTC的情况？ICH M7根据风险级别实施分类治理:警示结构单元-基因毒性杂质识别的起点警示结构单元：杂质结构中的某些具有与遗传物质发生化学反响水平特殊基团或亚单位.警示结构对于杂质的遗传毒性和致癌性具有重要提示作用：连接工艺限制、分析检测、毒理学评估的纽带.提升筛选效率,较少毒理学评估的工作量.在缺乏杂质平安性数据支持的情况下,EMA、FDA、ICH相关指导原那么中均将警示结构作为区分普通杂质和潜在遗传毒性杂质的主要指标. 常见警示结构单元模型：非遗传毒性致癌物的警示结构ICH M7限度限制的根本策略1〕、PDE 法适用于有阈值效应的遗传毒性杂质,即超过一定限度时才会产生遗传毒性的 杂质；限度确定参照残留溶剂限度的计算方法,根据相关动物的无可见效应剂量 〔NOEL 〕计算其可接受的日暴露量〔PDE 〕,再根据药品的最大日剂量计算出杂 质的接受限度.NOEL X 重量调整因子 Fl X F2 X F3 X F4 X F5PDE:允许的日暴露量〔mg/天〕 NOEL:无毒性反响剂量F1F1:物种之间的外推法因子〔F1:狗为2,小鼠为12,其他物种为10〕 F2:个体差异因子F3:毒性研究的周期因子 F4:严重毒性的因子F5:如果只有LOEL 〔最低无毒性反响剂量〕时的因子2〕、TTC 法对于无阈值效应的遗传毒性杂质,引入毒理学相关阈值〔TTC 〕的概念：假 设患者终生用药的癌症发生率不超过 10万分之一,从高浓度下进行的致癌性试 验数据线性外推到极低浓度得到的一个理论值.此类杂质,如果每日摄入量低于1.5 pg 那么患者因服药导致癌症发生的额 外风险,可以忽略不计.TTC 是一个风险治理工具,采用的是概率的方法：假设有一个潜在的遗传 毒性的杂质,并且我们对它的毒性不太了解,如果它的每日摄入量低于 TTC 值 〔1.5小前〕,那么它的致癌风险将不会高于10-5的概率.TTC 不能被理解为绝对无风险的保证.3〕、引入“短于终生〞 LTL 〔Less-than-lifetime 〕限度：基于TTC 可接受的限度为1.5仙g/day 假设患者终生服药的根底上得到的理 论值,在LTL 遗传毒性杂质限度的范围为：1.5 g g/day 365daysX 70years 〔15550 days 〕 =38.3mg 设计出平均分配在累计暴露量天数中的方法, 对于临床研究和上市药物不同 用药时间可接受的限度〔摄入量〕：4〕、含有多个遗传毒性杂质的限度要求：基于TTC 原理,当API 中含有不超过两个2类或3类遗传毒性杂质时,分 别单个限制,超过2个时,根据下表所述进行总量限制.PDE =需单独限制.1类以及API中的降解物需单独限制,不计入总限度.5〕、明确了需高度关注的危险物质限度原那么：类黄曲霉素aflatoxin-like-、N-亚硝基N-nitroso-化合物和氧化偶氮结构azoxy structures 致癌性明确,可能会显示出非常高的诱变性,可接受摄入量应显著低于M7定义的可接受摄入量,TTC 1.5 g g/day不适用于这类物质.可以应用M7的原理,具体问题具体分析,调整药物研发和上市药品中的可接受摄入量.如已有近似的相关结构的基因致癌数据的方法来判断研发和上市药品中相应杂质的可接受摄入量.ICH M7 : API的四种限制策略1、在最终API标准中采用适合的方法对相关杂质进行检测,进行常规限制, 到达适用的“短于终生〞〔LTL〕限度〔终产品限制〕根据ICH Q6A ,如果API中的遗传毒性杂质在至少6个连续的中试批次或3 个连续的生成批次中,测得结果均低于可接受限度的30%,那么可以论述进行定期检测；如果不满足该条件,那么建议对原料进行常规检测.2、对适宜的中间体中遗传毒性化合物的常规限制：采用适宜的方法对原材料,起始物料或中间体的相关杂质进行检测, 或进行生产过程的限制,同时制定可接受标准,使API杂质到达适宜的LTL限度.〔过程限制+终产品限制〕3、基于对工艺的熟悉,采用适宜的分析方法对原材料,起始物料或者中间体的杂质进行检测,或进行生产过程限制,同时确认这种限制方法能够保证API 杂质水平在可接受限度以下.〔过程限制〕①对适宜中间体中遗传毒性化合物的常规限制, 且限度＞适用的LTL限度；②充分的杂质危害性知识及去处和去除知识；③拟定工艺能使最终API杂质减少至30%适用LTL-限度.以上条件均符合时方可采用4、全面深入的工艺理解〔包括杂质危害性及去处和去除知识〕,基于科学的风险评估,以取代遗传毒性化合物的标准限制.①特别适用于不稳定〔如亚硫酰氯〕以及合成路线早期引入,但已被有效消除的杂质；工艺后期引入的那么需要提供与工艺相关的数据来充分论证该策略的合理性；②根据对杂质去向和消除产生影响的理化特性和工艺因素,包括化学反响性、溶解性、挥发性、离解性和所有用于去除杂质的物理处理步骤进行风险评估, 评估结果可以用来作为杂质被工艺所消除的预估因子.限制策略的相关考虑1、采用选项4,如果与选项3 一样仅根据科学原理来进行论述是不够充分的,还需要提交相关分析数据来支持限制方法.①后续化学反响中杂质的结构变化〔去向〕②中试批次分析数据.可根据实验室级别研究中的加标研究,来说明该杂质的去向/去除论证是严谨的,能够持续地保证杂质在最终API中残留量超过可接受限度的可能性可以忽略.③如果用于研发阶段的小规模模型被认为不能代表商业规模,那么一般需要确认中试规模和/或初始商业批次中所用的限制是适当的.2、假设选项3或4得不到证实的话,那么需要采用选项1或2.对于在较后合成步骤中引入的杂质,一般应采用选项1.3、对于已经定性为遗传毒性的潜在降解杂质,须说明该降解途径是否与原料药和制剂的生产工艺和/或其所拟的包装和存贮条件相关.①建议采用设计良好的加速稳定性试验〔如40C/75%, 6个月〕,采用拟定包装形式、适当分析方法来确定潜在降解产物的相关性.②或采用设计良好的动力学等效时间更短温度更高的稳定性试验,来针对拟定商业包装进行试验,在开始长期稳定性试验前确定降解途径相关性.③对于了解在降解途径,及产品中尚未发现的潜在降解产物的相关性尤其重要.4、假设所潜在降解产物在所拟包装和存贮条件下形成,且接近可接受限度, 那么应采取举措限制降解产物的形成. 并应监控采用所拟存贮条件、商业包装下长期稳定性试验中原料药或制剂的质量,并在质量标准中限制该杂质.EMA/CHMP金属催化剂或金属试剂残留量限度规定指导原那么1〕、按风险级别分类治理①第1类金属：具有显著平安性担忧.或疑心的致癌性其他显著毒性.1A 亚组：Pt、Pd1B 亚组：卜、Rh、Ru、Os1C亚组：Mo、Ni、Cr、V②第2类金属：具有低的平安性担忧.潜在的较低毒性. Cu、Mn等.③第3类金属：平安性担忧最小.无明显毒性. Fe Zn等.2〕、限度限制允许日接触量〔permitted exposure PDE〕浓度限度〔Concentration Limints〕浓度限度〔ppm〕 =PDE 〔小跃〕/每日给药量〔g/天〕**所列金属残留总和不得超过指定的限度分析方法1〕、适宜的、经验证的、专属性的方法①金属残留的形式可能不同于催化剂和试剂得初始形式②公认的药典方法或其他适宜的方法③仅有第2类或第3类金属,可采用非专属性的方法④对于1B亚组金属,可以接受0.5ppm的检测限.2〕、ChP重金属检查法-半定量测定方法,通常不适用于金属的定量测定.案例：某注射剂原料药制剂工艺用到了无水钳/碳催化剂,钳的PDE 10仙g 或浓度限度1ppm,本品采用ChP中检所检查法,限度10ppm,且专属性差,难以有效限制钳残留量.总结1、杂质研究与限制在药品研发中风险限制的关键环节之一：药品中杂质是否能得到合理、有效的限制,直接关系到药品的平安性.2、杂质研究与限制不是一项单纯的分析工艺,需要工艺、结构、分析、制剂、药理毒理等专业领域的协同联动,方可从根本上有效限制杂质.3、遗传毒性杂质、金属杂质的限制是一个新课题,其限制策略给业界和监管部门都带来了新的挑战.。

基因毒性杂质试题1)基因毒性杂质的控制合理限度：(1)<0.1% (2)<0.05% (3)<TTC 4)其他：2)Threshold of Toxicological Concern (TTC)的方法。

一个“ug/day”的TTC值，定量风险评估法：数据来源于大鼠致癌性分析，采用的风险概率为(一生即70 年暴露于该剂量杂质下，每万人有1 人死于癌症)。

3)按立卷审查要求基因毒性杂质研究策略为并订入研究未订入标准缺陷不研究不订入是缺陷4）有些结构基团具有较高致癌性，因此即使摄入量低于TTC水平，从理论上来说仍会导致可能的显著癌症风险。

这类具有较高致癌性的基团被称为“关注队列”，包括1、 2、3、 4、。

不适用TTC原则进行控制，需严控。

5）适用于基因毒性杂质研究法规：（1）ICH M7 (2)化学药物杂质研究技术指导原则（3）ICH Q3答案6）含基因毒性警示结构的：①酰氯R 应是除任何原子/基团②烷基苯磺酸酯R=C<的烷基包括卤代烷基，R1=除-OH-SH-O—S-之外的任何原子/基团④N-氧化芳香化合物应是任何芳香环或环2）有相同作用机制、结构相似的杂质，其含量的限度应该参考1.5微克/天的限量进行评估8）基因毒性杂质QBD策略（1）对于已知基因遗传毒性致癌物，首先考虑工艺中去除杂质，如除不去，则需进一步进行风险评估，如果通不过风险评估或风险无法估计，则应根据分期确定杂质阈值。

（2）已知基因毒性而致癌性未知的杂质,可根据风险程度，选择控制或者TTC 阈值控制。

（3）具有警示结构但与药物结构无关的基因毒性杂质，如无性数据确认无毒或实验无法证实无毒，则归入分类2杂质，按2类进行控制；如果DNA实验证实无毒，则按一般杂质控制策略进行控制。

（4）对于第四类具有警示结构且药物结构相关的遗传毒性杂质，可直接看药品是否有基因毒性，药品无毒按一般杂质策略进行控制，药品有毒，则要对药品上市的合理性进行再评估。

A rationale for determining,testing,and controlling specific impurities in pharmaceuticals that possess potential for genotoxicity Lutz Mu¨ller a,*,Robert J.Mauthe b,Christopher M.Riley c,Marta M.Andino d,David De Antonis d,Chris Beels e,Joseph DeGeorge f,Alfons G.M.De Knaep g, Dean Ellison f,Jane A.Fagerland h,Rebecca Frank i,Betsy Fritschel j,Sheila Galloway f, Ernie Harpur k,Charles D.N.Humfrey l,Alexander S.Jacks i,Nirdosh Jagota m, John Mackinnon e,Ganapathy Mohan k,Daniel K.Ness n,Michael R.O’Donovan l,Mark D.Smith o,Gopi Vudathala k,Larry Yotti pa Hoffmann-La Roche,PRBN-T,Bldg.73/311B,CH-4070,Basel,Switzerlandb Pfizer,Inc.,2800Plymouth Road,Ann Arbor,MI48105,USAc ALZA Corporation,1010Joaquin Road,Mountain View,CA94043,USAd Pfizer Inc.,Groton/New London Laboratories,Eastern Point Road,Groton,CT06340,USAe GlaxoSmithKline R&D Stevenage,Herts SG12NY,UKf Merck&Co.P.O.Box4,West Point,PA19486,USAg Johnson&Johnson Chemical and Pharmaceutical Development,Janssen Pharmaceutica,Turnhoutseweg30,B-2340Beerse,Belgiumh Abbott Laboratories,200Abbott Park Road,Dep.R45M,Bldg.AP31,Abbott Park,IL60064-6202,USAi Noramco,Inc.,1440Olympic Drive,Athens,GA30601,USAj Johnson&Johnson,410George Street,New Brunswick,NJ08901,USAk Sanofi-Aventis,11Great Valley Parkway,Malvern,PA19355,USAl AstraZeneca R&D,Alderley Park,Maccesfield,Cheshire,Cheshire SK104TG,UKm Wyeth Research,500Arcola Road,Collegeville,PA19426,USAn Eli Lilly and Company,Lilly Research Laboratories,2001W.Main St.,POB708,Greenfield,IN46140,USAo GlaxoSmithKline R&D,King of Prussia,Philadelphia,PA19406,USAp Bristol-Myers Squibb,Pharmaceutical Research Institute,Syracuse,NY13221,USAReceived29September2005Available online18January2006AbstractThe synthesis of pharmaceutical products frequently involves the use of reactive reagents and the formation of intermediates and by-products.Low levels of some of these may be present in thefinal drug substance and drug product as impurities.Such chemically reactive impurities may have at the same time the potential for unwanted toxicities including genotoxicity and carcinogenicity and hence can have an impact on product risk assessment.This paper outlines a procedure for testing,classification,qualification,toxicological risk assessment, and control of impurities possessing genotoxic potential in pharmaceutical products.Referencing accepted principles of cancer risk assess-ment,this document proposes a staged threshold of toxicological concern(TTC)approach for the intake of genotoxic impurities over var-ious periods of exposure.This staged TTC is based on knowledge about tumorigenic potency of a wide range of genotoxic carcinogens and can be used for genotoxic compounds,for which cancer data are limited or not available.The delineated acceptable daily intake values of between$1.5l g/day for$lifetime intake and$120l g/day for61month are virtually safe doses.Based on sound scientific reasoning,these virtually safe intake values do not pose an unacceptable risk to either human volunteers or patients at any stage of clinical development and marketing of a pharmaceutical product.The intake levels are estimated to give an excess cancer risk of1in100,000to1in a million over a lifetime,and are extremely conservative given the current lifetime cancer risk in the population of over1in4(/Regulatory Toxicology and Pharmacology44(2006)198–211/locate/yrtph0273-2300/$-see front matterÓ2005Elsevier Inc.All rights reserved.doi:10.1016/j.yrtph.2005.12.001*Corresponding author.E-mail address:Lutz.Mueller@(L.Mu¨ller).statfacts/html/all.html).The proposals in this document apply to all clinical routes of administration and to compounds at all stages of clin-ical development.It is important to note that certain types of products,such as those for life-threatening indications for which there are no safer alternatives,allow for special considerations using adaptations of the principles outlined in this paper.Ó2005Elsevier Inc.All rights reserved.Keywords:Pharmaceuticals;Impurities;Genotoxic;Carcinogenic;Risk assessment;Regulation;Staged TTC concept1.IntroductionResidual impurities resulting from manufacturing and formulation,or from degradation of the active pharmaceuti-cal ingredient(API)1and excipients,may be present in phar-maceutical products.A subset of these impurities may present a potential for genotoxicity and therefore pose an additional safety concern to clinical subjects and patients.The pharmaceutical industry and those that regulate it recognize their respective obligation to limit genotoxic impurities.Therefore,substantial efforts are made during development to control all impurities at safe concentra-tions.However,the effort made to limit impurities must be commensurate with the risk assessed at each phase of clinical development,taking into account the extent of the hazard,the disease indication,the size and characteris-tics of the exposed population,and the duration of that exposure,as well as the likely delay in the availability of beneficial medicines if the burden of limiting or controlling impurity levels is disproportionate.A balance of these con-siderations can be described best as the‘‘as low as reason-ably practicable’’(ALARP)2principle.It follows that the presence of impurities with genotoxic (mutagenic3)potential may be unavoidable in clinical trial and ultimately in approved and marketed materials.Con-trol of impurities in the drug substance and degradants in drug product are addressed in ICH Quality Guidelines Q3A(R)and Q3B(R),respectively,and the Q3C guideline that deals with residual solvents.However,no specific guidance for determining acceptable levels for genotoxic impurities is provided in these documents other than to rec-ognize the fact that unusually toxic impurities may require tighter limits of control.Toxicological assessment and jus-tifications of limits per these ICH guidelines are normally based on the qualification of representative batches of the API including its impurities in pivotal toxicity studies that include genetic toxicology tests.The European Medicines Agency Committee for Medicinal Products for Human Use(CHMP)has issued a Draft Guideline on the Limits of Genotoxic Impurities,which describes an approach for assessing genotoxic impurities of unknown carcinogenic potential or potency based on the TTC4concept(CHMP, 2004).The proposals detailed in this paper extend the CHMP approach to include the concept of a staged TTC that establishes allowable daily intakes of impurities based upon duration of exposure.It should be noted,however, that the CHMP draft document attempts to provide guid-ance to industry on how to address specifications for impu-rities possessing genotoxic potential in marketing applications for new drug products and does not consider how such impurities should be handled in the exploratory stage of drug development,i.e.,for clinical trial materials.This paper describes a process for testing,classifying, and controlling of such impurities in a way that balances therapeutic benefit with the potential risks associated with a medicinal product and concomitant levels of potentially mutagenic impurities.The process seeks to establish rational acceptance criteria that take into account the stage of clinical development,the duration of a clinical trial,sub-ject safety,and the feasibility of adequately sensitive ana-lytical methods.In the early stages of clinical development process and impurity information is limited and hence the emphasis is placed on known reagents,inter-mediates,and reaction products in the synthetic process. Both structurally identified impurities(those for which the chemical structure is known)and readily predicted impuri-ties(those that a technical review of the synthetic process1Abbreviations used:ADI,allowable daily intake;ALARA,as low as reasonably achievable;ALARP,as low as reasonably practicable;API, active pharmaceutical ingredient(note:API and DS—drug substance are synonymous and often used interchangeably);COC,cohort of concern; chemical groups requiring control to levels lower than the TTC due to potent carcinogenic potential;MTD,maximum tolerated dose;PDE, permitted daily exposure;Qualification—process of acquiring and evalu-ating data that establishes the biological safety of an individual impurity or a given impurity profile at specified levels(Draft CHMP Guideline); TTC,Threshold of Toxicological Concern;VSD,virtually safe dose.2Often,the As Low As Reasonably Achievable(ALARA)principle is referred to in product quality.This concept indicates that the detection and limitation of impurities at low levels is technically feasible with state of the art process and analytical technologies.However,the investment required to develop these capabilities for each candidate,particularly when the attrition rate of candidate molecules in early clinical trials is high,must be balanced with the need to control impurities to levels that are considered safe,pragmatic and practical.Thus,a distinction is made between ALARA levels that imply controlling levels to the lowest detectable level and ALARP levels that imply controlling levels to a practicable and safe level using the methods described in this paper.3The idioms‘‘genotoxic’’and‘‘mutagenic’’are used interchangeably in this publication.However,it is noted that they are not synonyms.While‘‘genotoxic’’very generally refers to any measurable DNA damage effects and includes,e.g.,indirect DNA damage measurements such as DNA strand breakage and DNA repair,the term‘‘mutagenic’’more specifically refers to heritable changes in DNA sequence or information content in somatic or germ cells.Such heritable changes are known to be important for critical steps in the process of carcinogenesis.4The Threshold of Toxicological Concern(TTC)was originally introduced by the FDA Center of Food Safety and Nutrition as a threshold of regulatory concern(i.e.,0.5ppb in the diet)as a level low enough to ensure that if an untested substance is later shown to be a potent carcinogen,the use of the substance would pose negligible concerns provided the regulatory criteria were met(Cheeseman et al.,1999).L.Mu¨ller et al./Regulatory Toxicology and Pharmacology44(2006)198–211199suggests might be present)are assessed and classified.After impurities are classified,acceptance criteria for impurity levels are set based on structural analysis,data from geno-toxicity testing,and by using a conservative risk-based approach based on the staged TTC.Since genotoxicity data are normally not suitable for a quantitative risk assessment,the(staged)TTC is based on animal carcinoge-nicity data and the knowledge about correlations between genotoxic processes and carcinogenesis for a substantial number of carcinogens.2.Classification of potential genotoxic impurities2.1.Considerations on testing of impurities for genotoxic potentialThe general framework for genotoxicity testing of phar-maceuticals is given in two internationally agreed ICH safety guidelines(ICH S2A,1995;ICH S2B,1997).One of these guidelines(ICH S2B,1997)describes the standard battery of tests for genotoxicity for drug substance,which consists of:(i)A test for gene mutation in bacteria.(ii)An in vitro test with cytogenetic evaluation of chro-mosomal damage in mammalian cells or an in vitro mouse lymphoma tk assay.(iii)An in vivo test for chromosomal damage in rodent hematopoietic cells.The ICH safety guidelines(S2A and S2B)state:‘‘For compounds giving negative results,the completion of this 3-test battery,performed and evaluated in accordance with current recommendations,will usually provide a sufficient level of safety to demonstrate the absence of genotoxic activity.’’In this context,genotoxicity is a broad term encompassing effects from mutagenicity through DNA reactivity,DNA damage,and chromosomal damage,both structural chromosome breakage and aneuploidy.Any compound that produces a positive result in one or more assays in the standard battery has historically been regard-ed as genotoxic,which may require further testing for risk assessment.Thus,the standard battery of genotoxicity assays used for testing the API provides important infor-mation about a diversity of mechanisms of genotoxicity, both directly and indirectly associated with effects on DNA(Mu¨ller et al.,1999).Genotoxicants that do not act directly on DNA are typically associated with threshold-re-lated mechanisms,while those that directly target DNA (typically detected in assays measuring the reverse or for-ward mutations in a specific gene with a selection agent) are considered by regulatory authorities not to have thresh-old-related mechanisms.Requirements for control of geno-toxic impurities in pharmaceutical products are different depending upon whether or not there is evidence for a threshold-related mechanism.DNA-reactive genotoxic impurities for which there is no evidence of a threshold-re-lated mechanism are regarded to be potentially trans-spe-cies and multi-organ carcinogens that may require control at relatively low levels.In contrast,it is accepted that impurities acting via threshold-related mechanisms do not require control at similarly low levels.Since the main concern that should drive control of impurities to rel-atively low levels is direct DNA reactivity,the primary end-point of relevance for genotoxic impurities is mutagenicity.Extensive knowledge about chemical functional groups that can react with DNA causing mutagenicity and concern regarding initiation of tumor processes is available in the scientific literature(Ashby and Tennant,1988,1991;Ashby and Paton,1993;Beningi,2004;Munro et al.,1996).Such knowledge has been used to develop rule-based computer programs such as DEREK(/ luk/derek/),MCase(/products/ prod01.htm),or TOPKAT(/ products/topkat/)and others.In addition,a recent analysis of the performance of various in vitro genotoxicity assays against the Carcinogenic Potency Database(CPDB) implies that a single mutation assay possesses the necessary sensitivity and specificity for detection of non-thresholded genotoxic carcinogenic chemicals(Kirkland et al.,2005). This has been confirmed using a larger database of carcin-ogens that includes proprietary data submitted to the US EPA and US FDA(Matthews et al.,2005).Hence, DNA-reactive carcinogens can be identified with a low inci-dence of false negative results by a procedure that com-bines the assessment of chemical structural features that infer DNA reactivity(such as electrophilicity)with a single biological hazard identification test such as a bacterial reverse mutation test,known as the‘‘Ames test’’(Bailey et al.,2005;Fetterman et al.,1997).Aflexible use of this approach is sometimes advisable since genotoxicity assess-ment of impurities in mammalian cells may be needed for specific structural groups,such as carbamates,which are known carcinogens and that are known to be inefficiently detected in bacterial genotoxicity tests(Allen et al.,1982).A clearly negative result in an appropriate genotoxicity test(i.e.,a bacterial reverse mutation test or mammalian cell assay)usually indicates a sufficient level of safety to conclude the absence of genotoxicity for the purpose of controlling impurities.2.2.Impurity classification with respect to genotoxic potentialIt is proposed here that impurities be classified into one offive classes using data(either published in the literature or from genotoxicity testing)and comparative structural analysis to identify chemical functional moieties correlated with mutagenicity.Thefive classes are:2.2.1.Class1—Impurities known to be both genotoxic (mutagenic)and carcinogenicThis group includes known animal carcinogens with reli-able data for a genotoxic mechanism and human200L.Mu¨ller et al./Regulatory Toxicology and Pharmacology44(2006)198–211carcinogens.Published data on the chemical structure exist demonstrating the genotoxic nature of the impurity.2.2.2.Class2—Impurities known to be genotoxic (mutagenic),but with unknown carcinogenic potential This group includes impurities with demonstrated muta-genicity based on testing of the impurity in conventional genotoxicity tests,but with unknown carcinogenic potential.2.2.3.Class3—Alerting structure,unrelated to the structure of the API and of unknown genotoxic(mutagenic)potential This group includes impurities with functional moieties that can be linked to genotoxicity based on structure,but which have not been tested as isolated compounds.They are identified based on chemistry and using knowledge-based expert systems for structure–activity relationships. The alerting functional moiety is not present in the structure of the parent API.Some widely recognized alerts for DNA reactivity,i.e.,mutagenic activity,are depicted in Fig.1.Some generic rule-based alerts may be quite unspecific (e.g.,the general alerts for aromatic amines;Cash et al., 2005);and further consideration must be given to chemical structural constraints,chemical environment,or experi-mental data in the assessment of potential genotoxicity. Due to the uncertain relevance of structural alerts,regula-tory action should not be based solely on the presence of a particular functional group;rather the accuracy for pre-dicted genotoxicity should be evaluated case-by-case based on the available scientific literature,additional unpublished (proprietary)data on the chemical class and further avail-able(genotoxicity)test results on closely related structures.2.2.4.Class4—Alerting structure,related to the APIThis group includes impurities that contain an alerting functional moiety that is shared with the parent structure. The genotoxicity of the isolated impurity is unknown,but the genotoxicity of the active principle has been character-ized through conventional genotoxicity testing.Similar chemical constraints and chemical environment exist for the alerting substructure in the impurity and the API.L.Mu¨ller et al./Regulatory Toxicology and Pharmacology44(2006)198–2112012.2.5.Class5—No alerting structure or sufficient evidence for absence of genotoxicityThis group would be adequately covered by existing ICH Q3A(R),Q3B(R),and Q3C guidelines.It has to be emphasized that this classification system would be used solely for the purpose to decide whether an impurity possesses a high level of risk and is therefore to be controlled at very low levels of daily intake.Hence, this classification is not a general classification of genotoxicity.3.Qualification of impuritiesThe relevant ICH guidelines concerning the qualifica-tion of impurities in commercial manufacture are Q3A(R)and Q3B(R)that focus on impurities in drug substances and drug products,respectively,while Q3C recommends limits for residual solvents in the drug prod-uct.The guidance given in these regulatory documents is considered to be applicable at the time of registration of a new pharmaceutical entity.Thefirst two guidelines describe threshold levels above which impurities are required to be reported,identified,and qualified either in toxicological investigations or in the clinic.The thresh-old levels vary according to the maximum daily dose of a drug.For drug substance,the identification thresholds are within the range of500and1000ppm(i.e.,0.05and 0.1%).ICH Guidelines Q3A(R)and Q3B(R)state that although identification of impurities is not generally nec-essary at levels less than or equal to the identification threshold,‘‘analytical procedures should be developed for those potential impurities that are expected to be unusually potent,producing toxic or pharmacological effects at a level not more than(6)the identification threshold.’’Thus in the case of impurities where a poten-tial safety concern for genotoxicity exists,the guidelines imply that the routine identification threshold is not con-sidered to be applicable.With regard to qualification,the requirements for qualifying potential genotoxic impurities are not specifically addressed in the guidelines and hence have been left to a case-by-case assessment.This case-by-case assessment is now proposed to be replaced by a general concept that is based on the knowl-edge and approaches as defined by the Threshold of Toxi-cological Concern(TTC).In agreement with the CHMP Draft Guideline on Genotoxic Impurities,the TTC concept (Barlow et al.,2001;Kroes et al.,2000;Kroes and Kozia-nowski,2002;Munro et al.,1999)is used to establish a limit of1.5l g/day as a virtually safe dose for most geno-toxic compounds,while recognizing that some highly potent genotoxic compounds(specifically N-nitroso com-pounds,azoxy-compounds,and aflatoxin-like compounds; see Fig.1for structural moieties)may require even lower levels(Kroes et al.,2004).Based on the conservative approach of linear back-extrapolation from animal cancer data,the TTC concept,despite some limitations,establish-es pragmatic limits for daily human exposure to genotoxic impurities assuming lifelong treatment(or intake as food contact materials;Bailey et al.,2005).Yet many medicines are given for limited time spans and to limited numbers of patients.Further,exploratory drugs are given in clinical development phases prior to marketing for limited dura-tion under well controlled conditions.Hence,a pragmatic approach should be appropriate for determining acceptable exposures to genotoxic impurities throughout clinical trials and for shorter-than-lifetime exposure.Based on the sto-chastic mode of action(dependency on total cumulative dose;Bos et al.,2004),the staged TTC approach outlined in Table1should be used to determine allowable daily lim-its for shorter-than-lifetime duration clinical studies.The conservative approach outlined in this paper regards all exposures>12months as potential lifetime exposures, unless specific arguments are given not to assume this.It is acknowledged that regulatory guidances for pharmaceu-ticals usually require rodent lifetime carcinogenicity tests when the clinical use(continuous or cumulative)of a phar-maceutical exceeds six months as this is referred to as ‘‘chronic’’for clinical use and thus requiring a lifetime ani-mal model for cancer risk assessment.However,it was felt that the use of the>6month intake criterion for ultimate control of genotoxic impurities at a calculatory lifetime cancer risk level would still be quite disconnected to theTable1Proposed allowable daily intake(l g/day)for genotoxic impurities of unknown carcinogenic potential during clinical development,a staged TTC approach depending on duration of exposure(ADIs for shorter durations than12months are based on linear extrapolation(Bos et al.,2004)from TTC value of 0.15l g/day(Cheeseman et al.,1999;Kroes et al.,2004))Duration of exposure61month>1–3month>3–6month>6–12month>12monthAllowable Daily Intake(l g/day)fordifferent duration of exposure(as normally used in clinical development)120a40a20a10a 1.5b or or or or0.5%c0.5%c0.5%c0.5%c c whichever is lower whichever is lower whichever is lower whichever is lowerKnown carcinogens should have compound-specific risk calculated(see text and Fig.1).a Probability of not exceeding a10À6risk is93%.b Probability of not exceeding a10À5risk is93%,which considers a70-year exposure.c Other limits(higher or lower)may be appropriate and the approaches used to identify,qualify,and control ordinary impurities during developed should be applied.In particular,approaches that foresee a very low dose of the API(‘‘microdoses’’)may facilitate higher limits than0.5%.202L.Mu¨ller et al./Regulatory Toxicology and Pharmacology44(2006)198–211basis for the cancer risk calculation,i.e.,a lifetime exposure of60–70years.Detection,quantitation,and control of potentially geno-toxic impurities to very low levels below the above men-tioned identification threshold presents considerable challenges for the synthetic and analytical chemist for the development,manufacture,and control of API,impurities in the API and the drug product.These challenges are most acute during the initial stages of development(pre-clinical, phase I and II)where manufacturing changes occur often so that evaluation of genotoxic impurities would likely have to be repeated,but still pertain later throughout development.Structural identification and characterization as well as robust control of impurities at low levels are gen-erally not achieved until the efficacy of the drug is estab-lished,a commercial route of synthesis is selected and a high level of process understanding is obtained.In particu-lar,for control of an impurity to a very low level,an under-standing of the functional relationship between process parameters and quality attributes learned through the syn-thesis of multiple lots is essential.Structural identification of a potential genotoxic impu-rity at a level above the identification threshold as per ICH guidelines,while sometimes still challenging,enables hazard identification and classification to be performed, followed by risk assessment and any necessary follow-up action.However,if such an impurity were present at a level below the identification threshold,unless there was a reason to be concerned a priori,the impurity is unlikely to be identified or quantitated,thus ruling out a formal risk assessment and the opportunity to apply subsequent control measures.Also,it is not possible to identify the structures of all possible impurities.Hence, for the purpose of controlling genotoxic impurities down to the level of the TTC concept,the ICH qualification of impurities needs to be adapted as laid out in the following proposal:Step1:Identify structural alerts in parent compound and expected impurities(both structurally identified and readily predicted),and classify the impurities into one of thefive classes defined above.Step2:Establish a qualification strategy for the impuri-ties based upon the classification,as detailed below.Step3:Establish acceptable limits of the impurity in the API,based upon the Allowable Daily Intake and the TTC concept.3.1.Step1:determination of structural alerts for parent compound and impurityPrior to the development of a qualification strategy,a scientific review of the synthetic route should be conducted to identify compounds of potential concern,including pro-cess impurities,reagents,or intermediates.All identified or readily predicted impurities are then classified into one of thefive classes described earlier.3.2.Step2:qualification strategy for impuritiesFollowing classification of the impurities,a qualification strategy defines the genotoxic potential of the impurity or establishes permitted specification limits for the impurity in the drug product.A summary of the qualification strat-egies based upon impurity classification is shown in Fig.2 and detailed below.3.2.1.Class1—Impurities known to be both genotoxic (mutagenic)and carcinogenicAn impurity may be identified as carcinogenic based on literature information and internal testing data.The pres-ence of a carcinogenic impurity is of highest concern to the safety of clinical populations.The goal in such situa-tions is to avoid these impurities by modification of formu-lation options or technology,synthetic route,starting materials,reactants,or purification steps.When it is not practical or realistic to avoid these impurities,specifica-tions should be determined as described below.If sufficient2-year rodent data are available to deter-mine the carcinogenic potency of the impurity itself,a com-pound-specific calculation of risk should be conducted. Different risk calculation methods are available,including the use of cancer slope factors,TD50values(the average daily dose estimated to halve the probability of remaining tumor-free throughout a2-year study),or maximum toler-ated dose(MTD)information(Gaylor and Gold,1995).If insufficient information is available to calculate a compound-specific risk,a general risk assessment based on the TTC concept can be employed.For genotoxic car-cinogens,this may be an exceptional case.Since the com-pound is in principle an established genotoxic carcinogen, Munro et al.(1999)derived0.3l g/day as the appropriate TTC,which is5-fold lower than the TTC for genotoxic compounds of unknown carcinogenic potential.Neverthe-less,the principles of the staged TTC should be used at out-lined in this paper.3.2.2.Class2—Impurities known to be genotoxic (mutagenic),but with unknown carcinogenic potential An impurity may be identified as genotoxic based upon the literature or by testing positive in one or more genotox-icity tests.Conventional genotoxicity tests are designed to identify genetic hazard,and the data generated in these tests are not suitable for quantitative characterization of risk.Thus,data from genotoxicity testing must be evaluat-ed for biological relevance,as some assays,i.e.,in particu-lar assays using mammalian cells in culture,are known to have a high rate of false positive results or poor correlation with rodent carcinogenicity(Kirkland et al.,2005;Mat-thews et al.,2005;Mu¨ller and Kasper,2000;Snyder and Green,2001).Impurities for which there is strong evidence of mutage-nicity,either from testing or from the literature,should be assessed on the basis of whether or not there is evidence for a threshold-mediated mechanism,as detailed below.L.Mu¨ller et al./Regulatory Toxicology and Pharmacology44(2006)198–211203。

基因毒性⼩册⼦基因毒性⼩册⼦Contents吗多明中吗啉的测定 (2)噻托溴铵中杂质离⼦的测定 (3)左⼄拉西坦中四丁基铵的测定 (4)盐酸法舒地尔中⾼哌嗪的含量测定 (5)苯甲酸阿格列汀中3-氨基哌啶的测定 (6)盐酸⽶诺环素中三⼄胺的测定 (7)坎地沙坦酯中羟胺的测定 (8)头孢曲松中甲基肼的测定 (9)重组⼈胰岛素中微量⼄醇胺的测定 (10)⽶氮平中⼆⼄基⼄醇胺的分析 (11)利培酮中羟胺、2-羟⼄基肼、肼的分析 (12)维格列汀中⾦刚烷胺的分析 (13)雷沙吉兰中羟胺的分析 (14)盐酸帕洛诺司琼中的（S）-（-）-3-氨基奎宁环胺双盐酸盐的分析 (15)考来维仑中有机胺类有关物质的分析 (16)安乃近中⼆甲氨基⼄醇的分析 (17)替加环素中叔丁胺的测定 (18)多肽中三氟⼄酸的测定 (19)疫苗中氰根的测定 (20)药物中叠氮根的测定 (21)药品中甲磺酸和三氟甲磺酸的测定 (22)吗多明中吗啉的测定1．简介吗多明是临床常⽤的预防⼼绞痛药物。

作为⼀种钙拮抗剂，吗多明可扩张⾎管平滑肌，减轻⼼脏⼯作负荷，减少⼼肌氧耗，且能扩张冠状动脉，促进侧枝循环，改善缺⾎⼼肌缺⾎。

在吗多明⽣产的起始原料中⽤到了吗啉。

吗啉是⼯业⽤重要环胺之⼀，由于其具有氮氧杂环的结构特点，在医药⽅⾯被⼴泛⽤于⽣产病毒灵、布洛芬、咳必定等多种药物的合成。

吗啉对黏膜、上呼吸道、眼睛、⽪肤等有强烈的刺激性，对⼈体有较⼤伤害，必须严格控其制成药中的残留量。

⽬前测定吗啉最常见的⽅法为⽓相⾊谱法，但由于吗啉极性较⼤，使⽤⽓相⾊谱法存在担体处理、⾊谱柱寿命等问题，且其灵敏度较低，⽆法满⾜质控要求。

吗啉的结构中含有伯胺，酸性条件下在阳离⼦交换⾊谱柱上保留良好，且在电导检测器上有⾮常灵敏的响应。

离⼦⾊谱电导法⽤于吗多明中吗啉的残留监测，结果准确可靠。

2. 实验部分Thermo Fisher Dionex ICS 5000+，AS-AP⾃动进样器；甲基磺酸（Fisher，99%），ThermoScientific Barnstead GenPure Pro超纯⽔；⾊谱柱：；IonPac CS16（5×250mm）保护柱：Ionpac? CG16（5×50 mm）；检测器：抑制电导检测；⾊谱图：图1 吗多明中吗啉的测定⾊谱图1-吗啉噻托溴铵中杂质离⼦的测定1．简介噻托溴铵由德国柏林格殷格翰公司研发，与辉瑞公司共同销售，2002年6⽉在荷兰和菲律宾⾸次上市。