A rationale for determining,testing,and controlling specific impurities in pharmaceuticals that possess p
otential 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 p
a Hoffmann-La Roche,PRBN-T,Bldg.73/311B,CH-4070,Basel,Switzerland
b Pfizer,Inc.,2800Plymouth Road,Ann Arbor,MI48105,USA
c ALZA Corporation,1010Joaquin Road,Mountain View,CA94043,USA
d Pfizer Inc.,Groton/New London Laboratories,Eastern Point Road,Groton,CT06340,USA
e GlaxoSmithKline R&D Stevenage,Herts SG12NY,UK
f Merck&Co.P.O.Box4,West Point,PA19486,USA
g Johnson&Johnson Chemical and Pharmaceutical Development,Janssen Pharmaceutica,Turnhoutseweg30,B-2340Beerse,Belgium
h Abbott Laboratories,200Abbott Park Road,Dep.R45M,Bldg.AP31,Abbott Park,IL60064-6202,USA
i Noramco,Inc.,1440Olympic Drive,Athens,GA30601,USA
j Johnson&Johnson,410George Street,New Brunswick,NJ08901,USA
k Sanofi-Aventis,11Great Valley Parkway,Malvern,PA19355,USA
l AstraZeneca R&D,Alderley Park,Maccesfield,Cheshire,Cheshire SK104TG,UK
m Wyeth Research,500Arcola Road,Collegeville,PA19426,USA
n Eli Lilly and Company,Lilly Research Laboratories,2001W.Main St.,POB708,Greenfield,IN46140,USA
o GlaxoSmithKline R&D,King of Prussia,Philadelphia,PA19406,USA
p Bristol-Myers Squibb,Pharmaceutical Research Institute,Syracuse,NY13221,USA
Received29September2005
Available online18January2006
Abstract
The 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$120l g/day for61month are virtually safe doses.Based on sound scientific reasoning,these virtually safe intake values do not pose an unacceptable risk to either human volunteers or patients at any stage of clinical development and mark
eting of a pharmaceutical product.The intake levels are estimated to give an excess cancer risk of1in100,000to1in a million over a lifetime,and are extremely conservative given the current lifetime cancer risk in the population of over1in4(
v/
Regulatory Toxicology and Pharmacology44(2006)198–211
www.elsevier/locate/yrtph
0273-2300/$-see front matterÓ2005Elsevier Inc.All rights reserved.
doi:10.ph.2005.12.001
*Corresponding author.
E-mail address:Lutz.Mueller@roche(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.
reactive substanceÓ2005Elsevier Inc.All rights reserved.
Keywords:Pharmaceuticals;Impurities;Genotoxic;Carcinogenic;Risk assessment;Regulation;Staged TTC concept
1.Introduction
Residual 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 gen
otoxic 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 ,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 identifi
ed impurities(those for which the chemical structure is known)and readily predicted impuri-ties(those that a technical review of the synthetic process
1Abbreviations 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 ,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 ,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)198–211199
suggests 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 impurities
2.1.Considerations on testing of impurities for genotoxic potential
The 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 gen
otoxic 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(www.chem.leeds.ac.uk/ luk/derek/),MCase(www.multicase/products/ prod01.htm),or TOPKAT(www.accelrys/ 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 submit
ted 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 ,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 potential
It 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 carcinogenic
This group includes known animal carcinogens with reli-able data for a genotoxic mechanism and human
200L.Mu¨ller et al./Regulatory Toxicology and Pharmacology44(2006)198–211
carcinogens.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 rel
ationships. The alerting functional moiety is not present in the structure of the parent API.Some widely recognized alerts for DNA ,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 API
This 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)198–211201
2.2.5.Class5—No alerting structure or sufficient evidence for absence of genotoxicity
This 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 impurities
The 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 identifica
tion thresholds are within the range ,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 o
n 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 the
Table1
Proposed 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 exposure
61month>1–3month>3–6month>6–12month>12month
Allowable Daily Intake(l g/day)for
different duration of exposure
(as normally used in clinical development)120a40a20a10a 1.5b or or or or
0.5%c0.5%c0.5%c0.5%c c whichever is lower whichever is lower whichever is lower whichever is lower
Known 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 an
d th
e approaches used to identify,qualify,and control ordinary impurities during developed should be applied.In particular,approaches that foresee a very low dose o
f the API(‘‘microdoses’’)may facilitate higher limits than0.5%.
202L.Mu¨ller et al./Regulatory Toxicology and Pharmacology44(2006)198–211
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