International Journal of Toxicology

http://ijt.sagepub.com/ Overview of Genotoxic Impurities in Pharmaceutical Development

Joel P. Bercu, Krista L. Dobo, Elmar Gocke and Timothy J. McGovern International Journal of Toxicology 2009 28: 468 DOI: 10.1177/1091581809349195 The online version of this article can be found at: http://ijt.sagepub.com/content/28/6/468

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Summary of Symposium
International Journal of Toxicology 28(6) 468-478 The Author(s) 2009 Reprints and permission: http://www. sagepub.com/journalsPermissions.nav DOI: 10.1177/1091581809349195 http://ijt.sagepub.com

Overview of Genotoxic Impurities in Pharmaceutical Development

Joel P. Bercu,1 Krista L. Dobo,2 Elmar Gocke,3 and Timothy J. McGovern4

Abstract This symposium focuses on the management of genotoxic impurities in the synthesis of pharmaceuticals. Recent developments in both Europe and United States require sponsors of new drug applications to develop processes to control the risks of potential genotoxic impurities. Genotoxic impurities represent a special case relative to the International Conference on Harmonisation Q3A/Q3B guidances, because genotoxicity tests used to qualify the drug substance may not be sufficient to demonstrate safety of a potentially genotoxic impurity. The default risk management approach for a genotoxic impurity is the threshold of toxicological concern unless a more specific risk characterization is appropriate. The symposium includes descriptions of industry examples where impurities are introduced and managed in the synthesis of a pharmaceutical. It includes recent regulatory developments such as the staged threshold of toxicological concern when administration is of short duration (eg, during clinical trials). Keywords genotoxic, impurities, drug substance, quality, ethyl methanesulfonate This symposium, chaired by Dr Angelique Braen and part of the American College of Toxicology (ACT) annual meeting, highlighted recent developments for risk assessment of genotoxic impurities. This is a challenging topic because risk assessments often have limited toxicity data sets, and coordination is required with expertise in different fields. Central to addressing these challenges is an understanding of the risks that may exist from a potential low-level exposure to a genotoxic and/or carcinogenic compound. This symposium provided (1) an overview on impurities in drug development, highlighting how genotoxic impurities fit in the current established guidances; (2) a recent example in which a threshold analysis of a genotoxic impurity was applied; (3) a description of some industry risk assessment practices and case studies of genotoxic and carcinogenic impurities; and (4) a discussion of recent European and US regulatory developments. controlling impurities in drug substances and drug products. Several regulatory guidance documents, including International Conference on Harmonisation (ICH) Q3A(R2), Q3B(R2), and Q3C(R4) provide recommendations on the identification, toxicological qualification. and derivation of allowable limits for drug substance impurities, drug product degradants, and solvents, respectively.1-3 For example, Table 1 illustrates ICH Q3A(R2) reporting, structural identification, and safety qualification thresholds for impurities in a drug substance. Similarly, ICH Q3B(R2) provides recommendations for the reporting, structural identification, and qualification of degradation products in new drug products. Stated thresholds in both guidances depend on the intended maximum daily dose of the drug. Although the ICH Q3 guidances do not provide guidance for controlling and assessing the safety of drug substance impurities during drug development because the stated intent of these documents is for marketing applications, it is important to exercise appropriate diligence starting with the initial clinical investigation. However, several considerations indicate application of somewhat less conservative identification and
1 2 3 4

Impurities and Drug Development: An Overview (Krista L. Dobo)

During the process of synthesizing active pharmaceutical ingredients (APIs), numerous starting materials, process intermediates, and reagents are used to arrive at the ultimate drug substance. Some of these materials, intermediates, and reagents, as well as by-products of synthetic processes, can have toxic properties and exist at low levels as impurities in the active ingredient or final drug product. If present at high enough concentrations, toxic impurities could elicit adverse health effects in humans. Therefore, pharmaceutical sponsors and regulatory authorities recognize the importance of
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Eli Lilly and Company, Indianapolis, IN, USA Pfizer Global Research and Development, Groton, CT, USA F. Hoffmann-La Roche Ltd, Basel, Switzerland SciLucent, LLC, Herndon, VA, USA

Corresponding author: Joel P. Bercu, Eli Lilly and Company, Lilly Research Laboratories, Health Safety and Environmental, Indianapolis, IN 46285, USA Email: jpbercu@lilly.com

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Table 1. Thresholds for Drug Substance Impurities Recommended in ICHQ3A(R) Maximum Daily Dose, g 2 >2 Reporting Threshold, % 0.05 0.03 Identification Threshold 0.1% or 1.0 mg/d (whichever is lower) 0.05% Qualification Threshold 0.15 or 1.0 mg/d (whichever is lower) 0.05%

qualification thresholds during the investigational stages of drug development. Early in drug development, clinical trials are small (ie, involving small cohorts of volunteers) and the duration of treatment is short. Both of these factors minimize the overall clinical risk. In addition, synthetic experience is limited and the analytical methods needed to identify and quantitate impurities are under development. Therefore, it is appropriate to apply higher identification and qualification thresholds during the early investigational phases with increased stringency as development progresses such that the sponsor is prepared to comply with ICH guidances upon registration. For example, although not specified in current guidances, an identification threshold of 0.2% and qualification threshold of 0.5% may be considered reasonable for early clinical investigations in which the maximum clinical doses do not exceed 1 g/d. However, cases in which high doses (several grams per day) are intended for the clinic may require more conservative limits. Likewise, investigations in patients with life-threatening indications or low-dose clinical investigations may allow for less conservative limits. The control and qualification of genotoxic impurities require special consideration given that some mechanisms of genotoxicity are assumed to have no discernable threshold and therefore any level of exposure may have an associated risk. ICH guidance documents do not provide specific recommendations for genotoxic impurities. However, the European Medicines Agency (EMEA) Committee for Medicinal Products for Human Use (CHMP) has published a guidance on limits of genotoxic impurities,4 and the US Food and Drug Administration (USFDA) has released a draft guidance as well.5 These guidance documents provide recommendations on allowable limits for genotoxic impurities for marketed products as well as for drug candidates during the clinical investigational phases. For genotoxic impurities for which a threshold mechanism is known, a permissible daily exposure (PDE) limit can be determined by using the methods outlined in ICH Q3C(R4). For genotoxic impurities for which a threshold mechanism is not known, the PDE is established using a threshold of toxicology concern (TTC). The TTC concept, adapted from an approach used for food contact materials, was developed as a screening tool to support conservative safe exposures when limited data are available.6,7 The TTC, when applied to genotoxic impurities, is based on a probability distribution of known carcinogens. It is a limit below which there will be negligible excess cancer risk, even if the genotoxic substance were to be a potent genotoxic carcinogen.6,8-10 Although conservative, the TTC can provide a toxicology cutoff even while limited information is known about the chemical. Both the EMEA and USFDA guidance documents recommend a TTC limit of 1.5

mg/d for marketed products (with some deviations being considered acceptable, eg, life-threatening indications) as well as staged-TTC limitshigher allowable daily limits for shorter durations of exposure during the investigational phases.5,11 However, although the TTC can be used as a default for genotoxic impurities, one should consider additional toxicity information, such as carcinogenicity data, which allow for a more precise risk analysis. Although diligence is necessary throughout clinical development and marketing to minimize exposure to genotoxic impurities, it is not practical or feasible to characterize every impurity present in the API down to very low limits. The earliest phases of clinical development are particularly challenging because a sponsor has limited experience with the synthetic processes (and associated profile of impurities) as well as the analytical methods necessary to identify and quantify impurities. Therefore, a pragmatic approach to identification of genotoxic impurities of concern is necessary in the early clinical development stages. One such approach involves evaluating all starting materials and intermediates used in the synthesis of an API for genotoxic potential via a structurebased assessment.12 For any starting materials or intermediates that are known or potential genotoxicants, an assessment of fate is conducted based on the conditions of synthesis to determine whether the substance will be purged or whether there is a potential for it to carry over to the API in significant quantities (ie, such that exposure would exceed staged-TTC limits). Any potentially genotoxic impurity for which there is not a high degree of confidence of removal during synthesis would then be a priority for analytical method development and process controls. Conduct of a structure-based assessment for the synthetic process used to make clinical supplies is recommended prior to the initial first-in-human investigation. In practice, this starts with the use of an in silico system (eg, DEREK) to identify substructures that are of concern for genotoxicity among starting materials, intermediates, and known or expected impurities. To further understand the genotoxic potential of alerting starting materials, intermediates, and impurities, additional due diligence can be conducted. That is, external databases (eg, TOXNET, SciFinder; VITIC) that are searchable by structure and substructure are mined to identify any genotoxicity and carcinogenicity data for the compounds of concern and/or structurally similar compounds. Structures of starting materials, intermediates, and impurities are also compared with the structure of the API. This is a particularly useful assessment for the penultimate precursor (or intermediates used late in the API synthesis). In many cases these intermediates are highly similar in structure to the API and may contain only alerting
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International Journal of Toxicology 28(6)

Table 2. Definitions of Impurity Classifications and Alignment of Classification With Approach to Control Human Exposure Impurity Classification Category 1 Category 2 Category 3 Category 4 Category 5 Definition Precedent for mutagenicity and carcinogenicity Mutagens with unknown carcinogenic potential or a close-in structural analog Alerting structureunique and unknown mutagenic potential Alerting structureNon-unique and qualified in comparison to API No structural alerting features Guidance on Control of Human Exposure PDE, TTC, or staged TTC TTC or staged TTC TTC, staged TTC, or test in Ames assay ICH Q3 controls apply ICH Q3 controls apply

PDE, permissible daily exposure; TTC, threshold of toxicological concern; ICH, International Conference on Harmonisation.

substructures in the same chemical context. The genotoxicity data generated for the API prior to the initiation of clinical investigation are very useful to understand the genotoxic potential of these intermediates. Ultimately, all of this information is taken into context in assigning each starting material, intermediate, and impurity a classification, as illustrated in Table 2.12,13 This genotoxic classification (Table 2) is a very effective means of communicating to synthetic chemists which potential impurities are of no concern and those which require additional consideration. Impurities classified as category 4 or 5 (alerting structure related to final API or no alerting structure) would be subject to control and safety qualification deemed appropriate for normal process-related impurities. In contrast, any impurities classified as category 1 or 2 (genotoxic carcinogens or mutagens) undergo a fate assessment based on the reaction conditions and synthetic processes that are used to make the API. When the outcome of the fate assessment indicates high confidence that the material will be removed and there is no evidence of its presence in the API based on existing analytical methods, there may be no further work prior to first-inhuman investigations. However, it is likely that a sponsor will commit to generating removal data at key synthetic steps. In cases where the outcome of the fate assessment suggests potential for the material to carry over into the final API, then this triggers the development of appropriately sensitive analytical methods. Depending on the outcome of the analytical assessment (ie, if the impurity is present at a level that is in excess or below the allowable TTC limit), additional work may be needed to purify the material prior to clinical investigations. For impurities that are classified as category 3 (structurally alerting but alerts have unknown relevance), there are a number of development options. One option is to assume the impurity is genotoxic and control the impurity accordingly (not to exceed the allowable TTC limit based on duration of treatment and highest clinical dose).5,11,13 There may be instances in which the synthetic processes and analytical methods already in place are sufficient to ensure that the API does not have levels of the impurity that would result in exceeding the staged TTC. Another option is to conduct an Ames test on the alerting impurity to determine whether it is mutagenic. This option would be more useful in circumstances in which the impurity is present at levels exceeding the staged TTC or in which
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analytical methods are not sensitive enough to confirm that exposure would be appropriately controlled. Positive results from the Ames assay would trigger the need to develop appropriately sensitive analytical methods and, potentially, depending on the outcome of analytical work, efforts to reduce the level of the impurity in the API. A negative outcome in the Ames assay would lead to the removal of the Genotoxic Impurity (GTI) risk classification and therefore application of control and qualification measures used for normal process-related impurities. In practical working experience, prioritization of measurement and control of genotoxic impurities in APIs require a multidisciplinary collaboration with experts in toxicology as well as synthetic and analytical chemistry. A toxicologist facilitates identification of structures of concern for genotoxicity and any subsequent risk assessment, the process chemist provides valuable insight regarding fate of concerning impurities in APIs, and the analytical chemist provides data (ie, level of impurity in the drug substance) necessary to conduct a human exposure assessment. This combined evaluation of genotoxic potential and fate of impurities in APIs provides a practical approach to focus on those impurities of greatest concern from the standpoint of hazard and exposure.

Thresholded Dose-Response Relationship for a Directly DNA Damaging Agent: Ethylmethane Sulfonate (Elmar Gocke)
The application of the TTC (ie, defaulted negligible limit for genotoxic compounds) approach to the qualification/specification of genotoxic impurities is based on a conservative linear back-extrapolation of carcinogenic effects determined at high and toxic dose levels to very low risk levels.4 A linear doseresponse relationship for DNA damage, mutation induction, and tumor formation is generally accepted as a sensible, precautionary assumption for compounds that directly damage DNA (one hit, one target), despite the evidence for existence of multiple layers of defenses that can lead to sublinear (upward concave) dose relations. Recently, evidence has been published on thresholded dose relations for induction of gene mutations and chromosomal damages in vitro14 for some of the most typical classes of directly acting genotoxinsnamely the

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1 0.9 0.8 % micronucleated PCE 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

EMS and ENU: micronucleus test in mouse bone marrow

50 45

Risk Assessment Based on Linear and Thresholded Dose-Response Relations

Following the currently accepted standard approach to risk assessment for a directly DNA-damaging genotoxin, initial risk calculations arrived at a worst-case prediction of less than 1 additional cancer in 10 000 Viracept patients exposed to the maximal dose of EMS (0.055 mg/kg/d) for the maximal period of 3 months. For the detailed assessment see Gocke et al.17 Although this estimate indicated that the human dose was close to the virtually safe dose considered as acceptable for genotoxic impurities based on the TTC approach (ie, 1 additional cancer in 100 000 patients), the publication of Doak et al14 suggested that additional studies could show evidence for a thresholded dose response for the genotoxic activity of EMS in vivo with the aim to reassure the patients that their exposure to EMS did not lead to any increase of the mutational load and by inference did not lead to any increase of cancer incidence above their background levels. In analogy to the in vitro studies by Doak et al, we included N-ethyl-N-nitrosourea (ENU) to differentiate the dose-response relations. Figure 1 shows that EMS-induced chromosomal damage with a clear threshold. Only at doses above 80 mg/kg/d was an increase of micronucleated polychromatic erythrocytes (PCE) seen. Statistical assessment confirmed a calculated threshold dose of 89.8 mg/kg/d. Figure 2 shows that a thresholded dose-response relationship was also evident for the induction of gene mutations by EMS. Calculated threshold doses were 35.4 mg/kg/d for bone marrow, 51.3 mg/kg/d for liver, and 24.5 mg/kg/d for gastrointestinal (GI) tract. In contrast, ENU induced effects with apparently linear dose relations. Already the lowest dose of 1.4 mg/kg/d induced clear increases above control. At the highest dose (22 mg/kg/d), mutation frequency (MF) values of more than 1000 10-6 were reached for bone marrow and GI tract and 450 10-6 for liver. In further experiments we could show that a single dose of 350 mg/kg of EMS induced an increase of the mutation frequency, whereas splitting this dose into 28 daily increments of 12.5 mg/kg did not induce an increase. In contrast, splitting the ENU dose did not lead to a decrease of the effectiveness.18 However, detailed analysis also suggests a threshold for ENU genotoxicity at a much lower dose level.17,19 Based on these studies, an unequivocal basis for a thresholded dose-response relationship for the genotoxicity of EMS could be established.20,21 Thus, it can be concluded that the patients were not exposed to an increased risk for genotoxic effects (and, therefore, carcinogenic and teratogenic effects)22 because the maximal human dose was lower by orders of magnitude than the lowest threshold dose. This conclusion was accepted by the European regulatory authorities.23 In the context of the general discussion on the TTC concept and is conservative basis, we can conclude that the default hypothesis of linear dose relations can be refuted for one of the most typical directly acting genotoxins. Instead of the TTC approach, the PDE approach (as outlined in the ICH
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EMS ENU

40 35 30 25 20 15 10 5 % of PCE

50

100 150 200 dose (mg/kg/ day)

250

0 300

Figure 1. Micronucleus test. Mice were treated for 7 days; bone marrow preparation was made on day 8. Solid lines, clastogenicity (% MN-PCE); dotted lines, bone marrow toxicity (% of PCE). EMS, ethylmethane sulfonate; PCE, polychromatic erythrocytes; ENU, N-ethyl-N-nitrosourea; MN, micronucleated.

alkylating agents ethylmethane sulfonate (EMS) and methylmethane sulfonate (MMS). Although the induction of DNA alkylations is shown to follow a linear, nonthresholded relationship,15 the repair of the lesions apparently is essentially error free up to a saturation level (ie, the threshold concentration). Although these in vitro data are suggestive, risk assessment for genotoxins should be based on in vivo data. Preferably, a threshold dose should be convincingly established by a comprehensive study program to serve as the basis for defining a PDE following the approach outlined in the EMEA guideline for genotoxins with threshold-related mechanisms.

The Case of EMS

Over a period of 3 months in 2007 (March to May), several thousand HIV patients in Europe were exposed to Viracept (nelfinavir mesylate) tablets containing the contaminant EMS.16 In early 2007, EMS had formed in a storage tank when the cleaning fluid (ethanol) was not properly removed and evaporated prior to refilling with methanesulfonic acid. The contaminant remained during the synthesis process and reached levels of approximately 1000 ppm in some batches of the finished product. Retrospective analysis showed that tablets produced prior to the incident had contained EMS at several orders of magnitude lower levels, thus verifying the initial measurements of representative batches during process development. Available in vitro and animal data did not permit a sufficiently convincing risk assessment for the levels at which HIV patients were exposed to EMS (maximal dose of 0.055 mg/kg/d). Therefore, the marketing authorization holder F. Hoffmann-La Roche conducted a series of preclinical investigations to better quantify the risk for adverse effects in the exposed individuals.

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International Journal of Toxicology 28(6) and pharmacokinetic studies shows no risk to the patients with a very large safety margin, as discussed above.

500 Mutant frequency (10-6) 400

EMS, ENU: LacZ mutations in MutaMouse

Conclusion
300 200 100 0

20

40 60 80 daily dose (mg/kg/day)

Liver EMS Liver addtl controls Liver ENU

100

120

Bone marrow EMS Bone marrow addtl controls Bone marrow ENU

GI tract EMS GI addtl tract controls GI tract ENU

Figure 2. Induction of LacZ gene mutations in MutaMouse. Mice were treated for 28 days; sampling was done on day 31. There were 7 animals per dose group. EMS, ethylmethane sulfonate; ENU, N-ethylN-nitrosourea; GI, gastrointestinal.

Unequivocal experimental evidence for a thresholded dose response was established for the genotoxicity of EMS. The in vivo studies corroborate similar evidence obtained from in vitro mutagenicity experiments.14 Although these findings cannot currently be extrapolated to other genotoxins, it has become obvious that the generally applied assumption of linear dose relations for mutagenicity (and by default carcinogenicity) for one of the most typical alkylating agents is not based on scientific fact. These studies support the use of risk extrapolation models different from the most conservative linear extrapolation even for typical genotoxins if experimental data (in vivo, but also in vitro) indicate sublinear dose-response relations. The TTC approach, based on linear extrapolation, should not be seen as being set in stone even for DNA-damaging genotoxins.

It is a regulatory expectation that genotoxic and carcinogenic impurities are minimized to negligible daily exposure levels.4,5,11,24 Risk assessment can provide a framework for identifying acceptable levels and the corresponding limits to ensure PDE NOEL weight adjustment=F1 F2 F3 F4 F5: patient safety from exposure to these impurities.13,25 The established risk assessment process involves hazard identification, When we use the most conservative uncertainty factors out- exposure assessment, dose-response assessment, and risk lined in ICHQ3C(R4), namely characterization.26 Hazard identification of impurities in a drug substance F1 12, for extrapolation from mice to humans (as based on involves an analysis of the synthetic scheme, including starting allometric scaling), materials, process intermediates, solvents, and reagents. F2 10, a factor to account for variability between individuals, Although published literature may contain genotoxicity or carF3 10, for studies of a shorter duration than 3 months, cinogencity data for known reagents or solvents, no toxicology F4 1 to 10, depending on the severity of the observed toxic data may be available for many novel compounds in the syneffects, and thetic scheme. An in silico or structure-based analysis can be F5 variable factor if the NOEL was not established (does not performed on all processing materials in the synthesis to deterapply for our assessment because a NOEL was mine potential genotoxicity.12 Any compounds predicted to be established, ie, F5 1), genotoxic can then be tested in a bacterial assay (eg, Ames) to prove or disprove the prediction.11 we calculate Exposure assessment evaluates the fate of an identified genotoxic or carcinogenic compound within the chemical synthPDE 25mg=kg=d 50 kg=12 10 10 10 1 esis of a drug substance. This understanding comes from 0:104mg=d: specific experience amassed as a drug substance moves through development. The closer a compound is introduced The value is about 70-fold higher than the TTC level of 1.5 mg/d to the final drug substance, the more of a concern it may be currently applied to EMS based on the generic linear back- as an impurity. For example, removal of genotoxic/carcinoextrapolation model for genotoxins acting via nonthreshold genic compounds that are introduced more than 4 steps back mechanisms. This PDE, derived with the use of very conserva- from the final processing step to negligible levels may be tive, generic uncertainty factors, is below the maximal dose demonstrated by an appropriate description of the process ingested by the patients. However, our detailed assessment22 chemistry.27 However, genotoxic/carcinogenic compounds based on genotoxicity, general toxicity, and drug metabolism introduced closer to the final processing step may require an
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Q3C(R4) guideline) could be followed for setting acceptable limits to EMS in drugs.3 A PDE is derived from the no observed effect level (NOEL) in the most relevant animal study. For the present assessment of EMS, the most relevant NOEL is taken from the MutaMouse study; thus NOEL 25 mg/kg/d.

Risk Assessment of Genotoxic and Carcinogenic Impurities: Current Issues and Case Studies (Joel P. Bercu)

Bercu et al
Table 3. Committee for Medicinal Products for Human Use23 Recommended Daily Exposure Limits for Clinical Development Duration of Exposure Single Dose Allowable daily intake, mg 120 1 mo 60 3 mo 20 6 mo 10 5

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12 mo

Table 4. US Food and Drug Administration5 Recommended Daily Exposure Limits (Draft) for Clinical Development Duration of Clinical Trial Exposure <14 d Genotoxic and carcinogenic impurity threshold, mg/d 120 14 d to 1 mo 60 1-3 mo 20 3-6 mo 10 6-12 mo 5 >12 mo 1.5

analytical strategy to show removal below toxicology-based safety limits. Dose-response assessment for a genotoxic or carcinogenic impurity in a drug substance depends on whether a threshold exists. If a threshold can be identified, the no observed adverse effect level (NOAEL) or lowest observed adverse effect level (LOAEL) is divided by appropriate safety factors to determine a PDE, as in the EMS example previously described.4 If no threshold can be derived, then linearity is assumed26 and the chosen risk assessment method depends on the available data. When adequate carcinogenicity data are available, risk assessment techniques might include the multistage model, benchmark dose, or TD50.28,29 When no carcinogenicity data are available, the TTC (ie, the dose of a genotoxic substance with a high probability of being less than a negligible cancer risk over background) is applied as a default.6,7,13 However, recent draft USFDA guidance allows for a risk assessment using comparator data for impurities without compound-specific carcinogenicity information when there is structural similarity to a known carcinogen.5 Risk characterization compares exposure of the impurity to the dose-response assessment. The basic assumption for linear extrapolation is that any dose can cause an effect, and therefore an acceptable exposure target is associated with a de minimis excess cancer risk. For pharmaceuticals, the typical acceptable excess cancer risk is 1 in 100 000.3,5,13 Because carcinogenicity risk is based on duration of exposure, the daily acceptable intake at the same excess cancer risk may be greater for shortterm versus chronic exposure.26,30 This principle allowed for acceptable higher daily exposures for short durations, for example, clinical trials (Tables 3 and 4).13 Extra conservatism (ie, an acceptable excess cancer risk of 1 in 1 million) is built in for short-term clinical trials because the population exposed generally includes volunteers who derive no benefit from the drug.5,11,13 Once the toxicology-derived limit is defined for the genotoxic impurity, then chemistry is designed such that the impurity is reduced below the limit. The following are examples based on experience with genotoxic and carcinogenic impurities:

Example 1
A compound was considered structurally alerting (aromatic amine) and was predicted to be positive in an Ames assay. The compound was then tested in the Ames and was determine to be genotoxic. The short-term TTC limit for phase 1 clinical trials (duration 30 days) was 60 mg/d (see Tables 3 and 4). Therefore, the toxicology-derived limit in the drug substance (maximum daily dose 1000 mg) was considered to be 60 ppm (60 mg per 1000 mg). An analytical test was performed with the aromatic amine, and it was not detected below the toxicologyderived limit. This was considered an acceptable scenario. When the drug substance was being further developed, a chronic TTC (1.5 mg/d) was applied and the toxicology-derived limit was lowered to 15 ppm based on a dose of 100 mg/d (1.5 mg per 100 mg). Analytically the impurity was shown to be below this limit, which was considered acceptable.

Example 2
Hydrazine was observed as a by-product reaction in a drug substance indicated for cancer. The maximum dose of the drug substance was 500 mg/d, and the duration was not more than 2 years. Based on the US Environmental Protection Agency (USEPA) cancer potency slope of 3/mg/kg/d, a lifetime exposure of 5876 mg was considered to be a 1 in 100 000 excess risk of cancer (http://www.epa.gov/iris/subst/0352.htm). Noncarcinogenic effects of hydrazine were evaluated as well, and it was determined that risk of cancer was the most sensitive end point.31 Therefore, based on the dose and duration of the drug substance, the acceptable daily dose for hydrazine was 8 mg/d (5876 mg/[365 2]). The resultant toxicology-derived limit was 16 ppm (8 mg per 500 mg).

Example 3
Studies with a drug substance under development in earlyphase lead optimization indicated that a genotoxic substance was formed as a degradation product in the stomach of test animals. Whether this was an impurity or a metabolite was questioned as part of an overall riskbenefit discussion for the
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474 molecule. Nevertheless, the product team decided to pursue a different molecule without the risk of a genotoxic substance being formed in the stomach.

International Journal of Toxicology 28(6) companies to remove these impurities to low (ppm) levels.27,33-39 The efforts to control genotoxic impurities in pharmaceuticals are not trivial, and some have questioned the overall public health benefits.40 Nevertheless, these measures are important because they ensure the quality and safety of the drug substance even when faced with uncertainty.

Example 4
t-Butyl chloride was formed as a by-product reaction in the synthesis of a new drug substance. The USEPA considers t-butyl chloride to be a group D compound or not classifiable as to human carcinogenicity (http://www.epa.gov/iris/subst/ 0417.htm) from available genotoxicity and carcinogenicity information. Based on the data available it was concluded that t-butyl chloride was not genotoxic. Therefore, under the ICH Q3A(R2) guidelines and available toxicity information, t-butyl chloride was considered a compound with unknown toxicity with a qualification threshold of 0.15%.1

Genotoxic Impurities: US and EU Regulatory Perspectives (Timothy J. McGovern)

Regulatory attention regarding genotoxic or carcinogenic impurities has increased in the last decade because of concern regarding their potential to induce genetic mutations, chromosomal breaks or rearrangements, and, ultimately, cancer. Although this increased regulatory attention may delay clinical development and drug product approvals, the safety of clinical trial subjects and patients is the primary concern of regulatory agencies.

Example 5
A synthetic scheme was being developed that had the potential to form several genotoxic impurities such as aniline compounds, and sulfonate esters. The synthetic scheme was redesigned to eliminate the potential for these genotoxic impurities.

Regulatory History
Until recently, the only available regulatory guidance addressing drug-related impurities included the ICH guidances Q3A(R2), Impurities in New Drug Substances, and Q3B(R2), Impurities in New Drug Products.1,2 These guidances address drug substance and drug product impurities, respectively, and set impurity qualification threshold limits based on a percentage of the drug substance or total daily intake. These guidances further state that certain qualification tests, including in vitro point mutation and chromosome aberration assays to assess genotoxic potential, can be conducted when these thresholds are exceeded. The studies can be conducted on the drug substance containing the impurity to be controlled, although studies using isolated impurities can sometimes be appropriate. The guidances also allow for higher or lower thresholds for qualification; lower thresholds can be appropriate if the impurity is unusually toxic, a classification considered relevant to genotoxic impurities. However, the guidances do not adequately address genotoxic impurities because they provide no clearly recommended approaches in the presence of positive results in genotoxicity assays, other than additional testing or reducing the impurity level, and do not specify what constitutes an acceptable level when an impurity is considered unusually toxic. Another ICH guidance, Impurities: Guidelines for Residual Solvents Q3C(R4), recommends limits for residual solvents.3 Not all recommended limits are directly based on genotoxic or carcinogenic potential; however, a limit of 2 ppm is recommended for benzene because of carcinogenicity concerns. The guidance recommends that only in cases where reliable carcinogenicity data are available should extrapolation by the use of mathematical models4,41 be applied to setting exposure limits. The increased regulatory attention, recognized potential for delays in drug development, and lack of specific regulatory guidance led to discussions at several scientific meetings and,

Future Issues
Risk assessment processes for genotoxic impurities should follow current regulatory guidances, but some areas are still being developed. For example, flexibility is allowed for life-threatening indications but there are no stated allowable daily exposure estimates.4 These estimates could depend on the indication (eg, Alzheimers disease vs cancer), exposure duration of the drug substance, and toxicity of the drug substance (eg, the drug substance itself is genotoxic). However, setting consistent limits will be difficult in these situations because a clear target is lacking. Guidances are also available for multiple genotoxic impurities.5,11 For structurally dissimilar compounds, separate limits are recommended whereas with structurally similar compounds the recommendation is to group exposure such that the combined limit will not exceed the TTC. This presumes that structurally similar compounds will act by a similar mode of action at the same organ site. However, it still is unclear at low doses as to whether structural similarity affects the overall risk assessment. Simulations of structurally similar impurities (defined as carcinogenic risks correlated with each other) versus dissimilar impurities (defined as carcinogenic risks independent of each other) indicate that structural similarity of multiple genotoxic impurities had minimal impact on overall carcinogenic risk.32 Nevertheless, it will be important to define structural similarity to address the potential additivity of multiple genotoxic impurities. Controlling genotoxic and carcinogenic impurities to low levels requires resources for the manufacture of pharmaceutical products because current risk assessment strategies for genotoxic and carcinogenic impurities are relatively conservative. Peer-reviewed publications in chemistry journals following the symposium show the recent focus applied by pharmaceutical
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Bercu et al eventually, the development of recommendations for handling genotoxic impurities by both regulators and industry. The EMEA CHMP released a draft guideline in 2004 that was finalized in 20064; documents were released in 2008 to address issues that arose following finalization of the guideline.11 The USFDA had not released any formal guidance at the time of this symposium, although USFDA representatives provided preliminary recommendations at various public meetings and in journal publications.24 The USFDA subsequently released a draft guidance document in December 2008.5 In addition, a Pharmaceutical Research and Manufacturing Association Task Force published a proposal in 2006.13

475 life expectancy (<5 years), treatment of a life-threatening condition, and other common exposures to the impurity can be used to justify a higher TTC level. The determination of the genotoxic potential of an impurity can be assessed by knowledge of existing genotoxicity/carcinogenicity data or by (Q)SAR analysis using knowledge-based systems (eg, DEREK, MultiCase). If no alerting features are identified, the impurity can be considered to be nongenotoxic. In the presence of an identified structural alert, the conduct of a bacterial reverse mutation assay (Ames), including either spiking with the impurity (minimum quantity of 250 mg per plate) or testing of the neat impurity, should be conducted. A negative Ames result would generally outweigh a (Q)SAR alert. If an alerting feature is identified but is shared by the API, the impurity may be qualified by negative genotoxicity data with the API. A staged-TTC approach to be applied during the various stages of clinical development was initially proposed by Muller et al.13 The CHMP considers this concept acceptable, and the recommended exposure levels, shown in Table 3, are extrapolated linearly from lifetime exposure data as suggested by Bos et al31; the values are generally 2-fold lower than those proposed by Muller et al to account for deviations from the linear extrapolation model. Regarding the control of multiple genotoxic impurities in a drug substance, if the impurities present different structural alerts they should be individually controlled at the TTC. Impurities with similar structural alerts should be controlled at the TTC based on the sum of the impurities because they could act by the same genotoxic mode of action and exert effects in an additive manner.

European Perspectives
The 2006 CHMP guideline provides recommendations for new applications to support approval of new molecular entities as well as existing active substances with new synthetic routes or other changes that may introduce new or higher levels of genotoxic impurities. A retrospective analysis of authorized products in the absence of a specific cause for concern is not recommended. The identification of genotoxic impurities should be a structure-based evaluation and should consider the full route of synthesis, including starting materials, intermediates, reagents, catalysts, and solvents. The evaluation of theoretical impurities arising from obvious side reactions based on existing knowledge should be considered. The primary focus of the CHMP guideline is on DNAreactive substances that have the potential for direct DNA damage. The guideline discriminates between genotoxic compounds with sufficient experimental evidence for a threshold-related mechanism and those without. For those with sufficient evidence of a threshold-related mechanism, an acceptable exposure level can be calculated as described for class 2 solvents in the Q3C Note for Guidance on Impurities: Residual Solvents.3 For those without sufficient evidence of a threshold-related mechanism, an acceptable limit based on a substance-specific calculation is possible if carcinogenicity data are available; a dose associated with a calculated risk level of 1 additional cancer death in 100 000 exposed for a lifetime is considered acceptable. In cases where a substance-specific calculation is not possible, the guideline recommends the use of a TTC, based on a probability distribution of carcinogenic potencies used to derive an estimate of a daily exposure level (mg per person) of most carcinogens that would give rise to less than a 1 in a million (1 10-6) upper bound lifetime risk of cancer (virtually safe dose). The CHMP guideline states, 1.5 mg/d intake of a genotoxic impurity is considered to be associated with an acceptable risk . . . for most pharmaceuticals. The TTC of 1.5 mg/d, corresponding to a 10-5 lifetime risk of cancer, is justified based on the benefits derived from pharmaceuticals. If the impurity level is limited to below the TTC, there is no need to apply controls based on the as low as reasonably practicable (ALARP) principle. There are special case compounds of high potency that are excluded from the TTC approach. Alternatively, certain factors, including short-term exposure, reduced

US Perspectives
As mentioned previously, a formal USFDA guidance regarding genotoxic impurities was not available at the time this symposium was held. Preliminary perspectives that were previously presented by USFDA representatives in other venues were covered in this session. The USFDA published draft guidance in December 2008. The USFDA guidance appears to share many similarities with the CHMP guideline, including the scope, recommended approaches to identify and deal with these impurities, and acceptable thresholds. The guidance intends to address identified and predicted impurities related to the API and synthetic processes that arise during clinical development and in activities supporting new marketing applications. The guidance also addresses impurities in supplemental NDA submissions in which novel impurities or relatively high levels of impurities are anticipated to occur through reformulations or the incorporation of new synthetic routes. As part of an initial impurity assessment, the observed impurity level should be compared with the qualification thresholds noted in the relevant ICH guidance documents; analytical methods to identify impurities of concern at appropriate levels are recommended. Once identified, the impurity should be evaluated for genotoxic potential based on the presence of
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476 alerting structures or known effects. If an alerting structure is present, the relationship of the impurity and the API in regard to the alerting structure should be considered; common alerting structures combined with negative genotoxicity results with the API would usually be sufficient to alleviate concern with the impurity. In the presence of a genotoxic impurity, the initial consideration should be to eliminate the impurity. It is recognized, however, that complete elimination is often not feasible and that the incorporation of new synthetic routes may result in new impurities of concern. An alternate approach is to implement process purification methods to reduce the impurity levels. Efforts should continue throughout development to further reduce the levels of concerning impurities to those associated with an insignificant increase in carcinogenic risk based on the given stage of clinical development. A variety of approaches can be taken to provide adequate safety qualification information for a potential or known genotoxic or carcinogenic impurity. When adequate carcinogenicity data are available, a compound-specific risk assessment can be conducted to determine an appropriate acceptance criterion; ICH Guidance Q3C appendix 3 recommends such an approach for class 1 carcinogenic solvents.3 In the absence of adequate carcinogenicity data, which will usually be the case, the results of appropriate in vitro genotoxicity assays should be considered. The assays ideally should be conducted with the isolated impurity to fully assess the genotoxic potential of the impurity. However, assays conducted with batches containing the impurity or the API spiked with impurity may support product specifications up to those tested. When the results of this testing present a potential concern, evidence of a threshold-related mechanism as discussed in the USFDAs 2006 guidance, Recommended Approaches to Integration of Genetic Toxicology Study Results,42 can be produced. Although the guidance was intended for evaluation of the API, the same considerations can be applied to an impurity. Alternatively, the impurity acceptance criterion can be reduced to a level associated with a daily intake at the TTC level of 1.5 mg/d, a level below which the exposure would not be expected to pose a significant carcinogenic or toxicologic risk, in order to support a marketing application. The USFDA also indicated that a staged-TTC approach is acceptable in supporting shorter term exposures during clinical development because clinical trials vary widely in duration, the TTC is based on risk estimates from lifetime rodent assays and the application of conservative assumptions, and there are recognized limitations in manufacturers ability to identify and control impurities during early development. At the time of this symposium, the specific acceptable levels for a staged threshold approach were not available. However, the recently released USFDA draft guidance includes recommended limits for clinical development (Table 4). Although the specific cutoff points for clinical exposure differ marginally, the overall approaches of the CHMP and the USFDA are similar. Structural activity relationship data can play a role in the overall safety evaluation of genotoxic impurities. This evaluation can include a review of information from the published
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International Journal of Toxicology 28(6) literature as well as in silico evaluations. Structural similarities to high- or low-potency compounds could indicate the appropriateness of lower or higher acceptable exposure limits to these impurities. Flexibility in the overall approach is allowable when appropriate. Consideration should be given to the stage of development (ie, marketing vs clinical development), the anticipated duration of treatment, the indication (late-stage cancer vs rhinitis), and similarities to compounds of known potency.

Case Study: Viracept

Dr Elmar Gocke previously discussed the risk assessment of EMS in Viracept tablets. In addition to this work, it is also interesting to note that the regulatory responses in the European Union and the United States differed. Although the product was initially recalled to the patient level in Europe and other affected regions, the recall did not directly affect the products status in the United States, Canada, or Japan. The primary reason for this difference appears to be the levels of EMS detected in the batches available for marketing in each region. Of note, Pfizer manufactures the drug batches for the US market, and EMS levels in batches produced by Pfizer were found to be substantially lower than those produced by Roche. In Europe, the European Community suspended the marketing authorization based on recommendations from the CHMP. Special concerns were identified for children and pregnant women, and it was concluded that there were insufficient data to establish a toxic dose to humans. A CHMP expert panel recommended the conduct of animal studies to identify a harmful level; an interim EMS specification of 0.6 ppm, corresponding to a maximum daily intake of approximately 1.5 mg, was proposed. In addition, the European Community recommended registries for certain populations. In the United States, the USFDA determined that most of Pfizers batches were within acceptable limits for EMS and recommended temporary limits to allow for continued use in populations where the benefits outweigh potential risks.43 Pfizer was asked to implement a new specification to limit the presence of EMS. The parties agreed on interim and long-term specifications; the interim specification was associated with an increased cancer risk in adults of less than 17 per 100 000 exposed and the long-term specification was associated with increased cancer risk in adults of less than 1 per 100 000.44 Dr Jeff Murray, the deputy director of the Division of AntiViral Products, stated that the difference in the response between the regions was a judgment call, because different agencies might think differently.45 According to Dr. Murray, the USFDA response was a riskbenefit decision and the agency decided that it was better for patients to be exposed to higher, interim levels for a short period of time rather than risk an abrupt medication switch or drug shortage. He also indicated that this may be a wide-ranging issue for drugs that have mesylate, besylate, or tosylate salts and that all of the products could come under the final recommended specification.

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Bercu et al Based on the conducted studies and risk assessment described by Dr Gocke, the EMEA released a question-andanswer document in July 200823 indicating that Roche completed in-depth studies that demonstrated the presence of a threshold effect for EMS and that patients exposed up to 0.05 mg/kg were not at an increased risk.

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8. Rulis AM. Establishing a threshold of regulation. In: Bonin JJ, Stevenson DE, eds. Risk Assessment in Setting National Priorities. New York, NY: Plenum; 1989:271-278. 9. Cheeseman MA, Machuga EJ, Bailey AB. A tiered approach to threshold of regulation. Food Chem Toxicol. 1999;37: 387-412. 10. Munro IC. Safety assessment procedures for indirect food additives: an overview. Report of a workshop. Regul Toxicol Pharmacol. 1990;12:2-12. 11. Committee for Medicinal Products for Human Use, European Medicines Agency. Question & answers on the CHMP Guideline on the Limits of Genotoxic Impurities. 2008. EMEA/CHMP/ SWP/431994/2007. http://www.emea.europa.eu/pdfs/human/swp/ 43199407en.pdf. Accessed September 25, 2009. 12. Dobo KL, Greene N, Cyr MO, et al. The application of structurebased assessment to support safety and chemistry diligence to manage genotoxic impurities in active pharmaceutical ingredients during drug development. Regul Toxicol Pharmacol. 2006;44:282-293. 13. Muller L, Mauthe RJ, Riley CM, et al. A rationale for determin ing, testing, and controlling specific impurities in pharmaceuticals that possess potential for genotoxicity. Regul Toxicol Pharmacol. 2006;44:198-211. 14. Doak SH, Jenkins GJ, Johnson GE, et al. Mechanistic influences for mutation induction curves after exposure to DNA-reactive carcinogens. Cancer Res. 2007;67:3904-3911. 15. Swenberg JA, Fryar-Tita E, Jeong YC, et al. Biomarkers in toxicology and risk assessment: informing critical dose-response relationships. Chem Res Toxicol. 2008;21:253-265. 16. Muller L, Gocke E, Lave T, et al. Ethyl methanesulfonate toxicity in Viracept: a comprehensive human risk assessment based on threshold data for genotoxicity. Toxicol Lett. In press. 17. Gocke E, Muller L, Pfister T. EMS in Viracept: initial (traditional) assessment of risk to patients based on linear dose response relations. Toxicol Lett. In press. 18. Gocke E, Ballantyne M, Whitwell J, et al. In vivo MNT and MutaTMmouse studies to define the dose response relations of the genotoxicity of EMS and ENU. Toxicol Lett. In press. 19. Johnson GE, Doak SH, Skibinski D, et al. Linear dose-response of DNA-reactive genotoxins: recommendations for analysis. Mutat Res. In press. 20. Gocke E, Wall M. In vivo genotoxicity of EMS: Statistical assessment of the dose response curves. Toxicol Lett. In press. 21. Lutz RW, Lutz WK. Statistical model to estimate a threshold dose and its confidence limits for the analysis of a sublinear dose response relationship, exemplified for mutagenicity data. Mutat Res. In press. 22. Muller L, Gocke E, Lave T, et al. Threshold risk assessment for ethyl methanesulfonate toxicity, including susceptibility differences regarding tissue, species and individual age and predisposition. Toxicol Lett. In press. 23. Committee for Medicinal Products for Human Use, European Medicines Agency. Questions and answers on the follow-up to the contamination of Viracept (nelfinavir) with ethyl mesilate. 2008. EMEA/CHMP/375807/2008. http://www.emea.europa.eu/ humandocs/PDFs/EPAR/Viracept/Q&A_Viracept_37580708en.pdf. Accessed September 25, 2009. 477

Conclusion
The European Union and United States share many similarities in their approaches regarding genotoxic impurities. It is recommended that these impurities be managed appropriately to levels anticipated to impart insignificant risk to subjects and that approaches apply good science to ensure safety without unnecessarily limiting drug development. Although regulators have provided some initial guidance regarding impurities with genotoxic potential, additional challenges continue to be identified as all parties gain more experience in this area and these challenges will need to be addressed. Therefore, the assessment and regulation of genotoxic impurities will likely evolve as technological and risk assessment approaches are further developed. Acknowledgements
We thank Dr Angelique Braen for chairing the symposium and facilitating this article. We also thank the American College of Toxicology for inviting us to speak in their annual meeting.

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