Overview of impurities in pharmaceuticals ...

Overview of impurities Asian Chemistry Letters in pharmaceuticals: Toxicological aspects

87 Vol. 16, No. 1 (2012)87-97

Overview of impurities in pharmaceuticals: Toxicological aspects Ismet Cok and Esra Emerce Gazi University, Faculty of Pharmacy, Department of Toxicology Hipodrom, 06330, Ankara, Turkey In drugs, the presence of unwanted chemicals regarded as impurity may influence the efficacy and safety of pharmaceutical products even in very small amounts. Since 1994, certain regulations relative to impurities in drug applications were intended to be set by international regulatory authorities such as ICH (International Conference on Harmonization), FDA (Food and Drug Administration), EMEA (European Medicines Agency), particularly, considering genotoxic impurities in pharmaceuticals at trace level which may play a role in causing mutagenesis and carcinogenesis in humans. Often, technically it is not possible to remove all impurities from the drugs during the production process. These elimination processes also increase the costs. In this context, risk assessment should be conducted in order to set a balance between the need to reduce the impurity concentration at the lowest possible level and the practical feasibility of this reduction. Therefore, a monitoring study for impurities that may occur from the production process through the usage and storage of drug is needed. The monitoring studies should cover determination and quantification of impurities together with assessment of their toxicological results. In the present day, there is still little knowledge about impurities and their toxic effects in literature. This review covers the details of impurities in pharmaceuticals, their regulations, toxicological aspects, and analytical strategies.©Anita Publications. All rights reserved.

1 Introduction Advancements in pharmaceuticals produced for consumption as well as clinical trials necessitate risk/ benefit assessment involving regulatory agencies, health care professionals. On the other hand, pharmaceuticals used in treatments also entail side effects which may be acceptable depending on the disease whether the disease is a life threatening condition or not. In the overall risk evaluation of the therapy, the other key parameter which will be taken into account is the duration of the treatment [1]. 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 can have toxic properties and exist at low levels as impurities in the active ingredient or final drug product [2]. Since these impurities convey only risk without any benefit, the overall risk of the therapy increases depending on number, type and concentration of these impurities. Drug impurities might be called as “pollutants” in the pharmaceutical world [3]. The safety of pharmaceutical product depends on the toxicological properties of the active drug substance, as well as the impurities character of being formed during the various chemical transformations. Therefore, identification, quantification, and control of impurities in the drug substance and drug product are important parts of drug development for acquiring marketing approval [4]. Therefore, pharmaceutical sponsors and regulatory authorities adhere importance to the control of impurities in pharmaceuticals [2]. An impurity can be defined as any component, together with the drug substance, or ingredients, arising out of synthesis, degradation, or unwanted chemicals that remains with API’s in the pharmaceutical world. The drug substance is compromised in terms of purity even if it contains another material with superior pharmacological or toxicological properties. Any extraneous material present in the drug substance has to be considered an impurity even if it is totally inert or if it has superior pharmacological properties [5]. Even in small amounts the existence of these unwanted chemicals, may affect the efficacy and safety of the drug products. Corresponding author : e-mail: [email protected]; Phone: 0090 312 202 30 86 (Prof Ismet Cok)

Overview of impurities in pharmaceuticals: Toxicological aspects

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According to International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines, impurities can be classified into the following categories [6]: Organic Impurities: Organic impurities may occur during the manufacturing process and/or storage of the drug substance and they may include starting materials, by-products, intermediates, degradation products, reagents, ligands and catalysts. Inorganic Impurities: Inorganic impurities may occur from the manufacturing process and include heavy metals, inorganic salts, miscellaneous (reagents, ligands, and catalysts) and other materials (filter aids, charcoal, etc.). Residual Solvents: Residual solvents in drug substances, excipients, and drug products may be generated during the drug production or they are volatile solvents are used during the manufacturing process. Since mostly it is not 100% possible to yield a single end product in synthetic organic chemistry, it is a strong probability to get by-products together with the targeted product. Impurities may be classified into several sources, such as; crystallization-related impurities, stereochemistry-related impurities, residual solvents, synthetic intermediates and by-products, formulation-related impurities, impurities arising during storage, method related impurity, mutual interaction amongst ingredients, and functional group-related typical degradation [7]. 2 Developments of regulations on impurities and their impacts Works on guidelines regarding the impurities in pharmaceuticals started in 1994 and continued with “Q3A(R2), Impurities In New Drug Substances”, “Q3B(R2), Impurities In New Drug Products”, “Q3C(R4), Impurities: Guideline for Residual Solvents”, “Q3D, Impurities: Guideline for Metal Impurities” which have been published until now by ICH [6, 8, 9, 10]. In addition to ICH guidelines, the U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER) released “Guidance for Industry ANDAs: Impurities in Drug Substances” [11]. These published guidelines provide information on impurities concerning reporting, identification, quantification and determination of threshold limits which do not reveal toxicological consequences in drug substances and products. Furthermore, they provide information and recommendations about safety assessments in new drug developments and registration applications for pharmaceutical manufacturers and relevant health authorities, as well as approaches on controlling and risk assessment of impurities. ICH Q3A(R2) addresses impurities in new drug substances produced by chemical syntheses and not previously registered in a region or member state while ICH Q3B(R2) guideline addresses only those impurities in new drug products classified as degradation products of the drug substance or reaction products of the drug substance with an excipient and/or immediate container closure system. They both do not apply to new drug substances/products used during the clinical research stages of development. According to ICH guidelines, in new drug substances consumed less than 2 g per day, if the impurity exists in new drug substance at a level greater than 0.05%, it should be reported, if it exists at a level greater than 0.1%, it should be identified, and if it exists at a level greater than %0.15, its qualification data should be provided. In the case where maximum daily dose is greater than 2 g, impurity thresholds for reporting, identification and qualification are 0.03%, 0.05%, and 0.05%, respectively. On the other hand, the thresholds of the impurities in new drug products are also described in terms of the amount of drug substance administered per day. However, it is noted that lower or higher threshold limits may be appropriate for some specific cases. Depending on the fact that impurity is unusually potent, producing toxic or pharmacological effects, then lower limits may be considered. But on a case-by-case basis assessment higher threshold limits may also be set provided that they are justified scientifically [6, 8].


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On the other hand, ICHQ3C(R4) guideline describes levels considered to be toxicologically acceptable for some residual solvents in pharmaceuticals and recommends use of less toxic solvents. Due to the fact that no therapeutic benefit is gained from residual solvents it is safe to assume that all residual solvents should be removed in order to reach product quality or other quality-based requirements. According to the guideline, residual solvents are classified into three groups depending on the possible risk to human health: “Class 1 solvents: Solvents to be avoided”, “Class 2 solvents: Solvents to be limited” and “Class 3 solvents: Solvents with low toxic potential”. Also the concept of “Permitted daily exposure” (PDE) is defined in the guideline as a pharmaceutically acceptable intake of residual solvents. The PDE is derived from the no observed exposure level (NOEL), or the lowest observed effect level (LOEL) in the most relevant animal studies using “uncertainty factors” which are used for extrapolating the dose from animals to humans and the individual variability [9]. The guideline on the metal impurities published by EMEA (European Medicines Agency) aims to recommend maximum acceptable concentration limits in pharmaceutical substances or in drug products for the residues of metal catalysts or metal reagents that may exist. This guideline classifies metal residues into three categories based on their individual level of safety concern and sets concentration limits. The limits are based on the maximal daily dose, duration of treatment, and administration route of the drug product as well as the permitted daily exposure (PDE) of the metal residue. Recommendations on testing strategies, analytical procedures and reporting levels in pharmaceutical substances or drug products are also included in the mentioned guideline [12]. In addition to the EMEA, ICH also proposed to develop another guideline with the purpose of drafting a global policy for limiting metal impurities qualitatively and quantitatively in drug products and ingredients [10]. On the other hand, the current United States Pharmacopeia (USP) test for metals is nonspecific and is not sufficiently sensitive to control highly toxic metals at levels that present health concerns. The USP Heavy Metals test variants are also the current standard in the European Pharmacopoeia 6.0 Chapter 2.4.8 “Heavy Metals”, the International Pharmacopoeia 4th Edition Chapter 2.2.3 “Limit Test for Heavy Metals”, and the Japanese Pharmacopoeia XV Chapter 1.07 “Heavy Metals Limit Test” [13]. Guidelines on Genotoxic Impurities Among the toxicological endpoints the carcinogenicity and mutagenicity are considered to pose the highest concern for human health and for this reason the object of extensive research activity, as well as of recognized regulatory testing methods [14]. Genotoxic substances are those which impact genetic material by means of mutations. Mutations may also occur indirectly by activating a cell to produce genotoxic substances. Because of this, it is important to identify genotoxic substances at very low levels to ensure public safety. Genotoxic impurities (GTIs) can induce genetic mutations, chromosomal breaks, and/or chromosomal rearrangements and have the potential to cause cancer in humans [15]. It is estimated that the intermediary steps within pharmaceutical development process may have 20–25% potentially genotoxic intermediates [16]. The limits which have been set for nongenotoxic impurities are not acceptable for GTIs due to their adverse effects and that they pose an additional safety concern to patients. Therefore, according to the daily dose of drug substance limits are needed to be set [15, 17]. ICHQ3 guidelines do not clarify how to handle GTIs and they do not provide specific recommendations for GTIs. With regard to what is express in the guidelines, it is only mentioned that “Lower thresholds may be appropriate for unusually toxic impurities”. Although it is thought that with the aforementioned statement it is referred to impurities that are genotoxic, GTIs which cause crucial concern do not take up a place aside from the recommendations on controlling of some carcinogenic solvents. Within this context, a draft position paper on the limits for GTIs was published by the Committee for Proprietary Medicinal Products (CPMP) on behalf of the EMEA in 2002. Later on, EMEA Committee for


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Medicinal Products for Human Use (CHMP) has finalized the work and published the “Guideline on the Limits of Genotoxic Impurities” which defines a general framework and practical approaches on how to deal with GTIs in new active substances and new applications for existing active substances as well. However, it does not need to be applied to already commercialized products if there is not a specific reasonable cause for concern. In order to determine the acceptable levels of exposure to genotoxic carcinogens, possible mechanisms of action and dose-response relationship are considered to be important components. The guideline suggests distinguishing GTIs into two groups based on the above considerations: a) Genotoxic compounds with sufficient (experimental) evidence for a threshold-related mechanism b) Genotoxic compounds without sufficient (experimental) evidence for a threshold-related mechanism. According to genotoxicity mechanisms, it is recommended to use different approaches in controlling GTIs. For the former, acceptable exposure levels of GTIs can be set calculating the PDE limit as described previously in the methods for residual solvents impurities. For the latter, the guideline recommended the control of levels of unavoidable GTIs according to the “as low as reasonably practicable” principle (ALARP principle) [18]. The foundation of the ALARP Principle is to set a balance need to reduce the impurity concentration at the lowest possible level and achieve technical feasibility of this reduction. In the assessment of the mentioned practical feasibility there should be the involvement of the evaluation of process capability to remove the impurity and also the development of suitable analytical methods for its quantification [1]. In the EMEA guideline, a threshold of toxicological concern (TTC) approach is described for assessing GTIs of unknown carcinogenic potential or potency. As a risk assessment tool, TTC is based on the principle of establishing a human exposure threshold value for all chemicals, below which there is a very low probability of an appreciable risk to human health. The concept is based on extrapolation of toxicity data from one or more available databases to a chemical compound for which the chemical structure is known, but for which no or limited toxicity data is available. From the perspective of consumers, industry and regulators the establishment and application of widely accepted TTC values would be beneficial. The TTC approach is expected to channel limited resources of time, cost, animal use and expertise to testing and evaluation of substances with higher potential risk to human health below the above mentioned the threshold and contributes to reduce animal use [19]. It should be known that the TTC concept is not implemented to carcinogens where adequate toxicity data (long-term studies) are available. However it enables a compound-specific risk assessment such as N-nitroso, azoxy, aflatoxin-like chemicals as high potency genotoxic carcinogens. In this co rived from pharmaceutical treatment compared to other areas which uses the same concept. Certain conditions such as short-term exposure periods may justify the determination of higher limits [18]. Afterwards, EMEA also published a “Question & Answers on the CHMP Guideline on the Limits of Genotoxic Impurities” in order to provide clarification and harmonization of the Guideline which was published in 2006 [20]. Current available guidelines recommend the use of the TTC for a single impurity where mutagenicity but no carcinogenicity information exists. Bercu et al. have discussed to characterize cancer risk for a mixture of GTIs by using a probabilistic analysis. They observed that cancer risk was increased in the presence of more than one genotoxic impurity in a new drug substance. However, considering the conservative assumptions of the TTC, this increase was assessed to be relatively small [21]. The EMEA Q&A document sets the limits when more than one GTI is present in the drug substance and only by having structurally unrelated impurities the TTC limit of 1.5 µg/ day can be applied to each individual impurity. When GTI are structurally related, the limitation of the sum of the exposures at 1.5 µg/person/ day is recommended. Pharmaceutical Research and Manufacturing Association (PhRMA) expert group, led by L. Muller, elaborated a “staged TTC” approach for the intake of GTIs over various durations of exposure. In this aspect, the TTC acceptable daily dose of 1.5 µg/ person became 120, 40, 20 or 10 µg/ person, respectively,


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depending on the increase of the duration of exposure from less than 1 month to 12 months [17]. This recommendation was accepted by EMEA though by taking into account a dose rate correction factor of 2. Thus, the acceptable limits for daily intake of GTIs are 5, 10, 20, [20]. The paper has classified impurities with respect to genotoxic potential: Class 1—Impurities known to be both genotoxic and carcinogenic; Class 2—Impurities known to be genotoxic, but with unknown carcinogenic potential; Class 3—Alerting structure, unrelated to the structure of the API and of unknown genotoxic potential; Class 4—Alerting structure, related to the API; Class 5—No alerting structure or sufficient evidence for absence of genotoxicity. After classification of impurities, a qualification strategy is provided and established acceptable limits of impurities in the API, based on the allowable daily intake and the TTC concept [17]. In 2008 Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) issued draft guideline for industry: ‘‘Genotoxic and Carcinogenic Impurities in Drug Substances and Products: Recommended Approaches’’. This guideline addresses recommendations on how to evaluate the safety of genotoxic and carcinogenic impurities during clinical development (investigational new drug applications (INDs)) and for marketing applications (new drug applications (NDAs), biologics license applications (BLAs), and abbreviated new drug applications (ANDAs)). There are significant similarities between the FDA draft guideline and the EMEA guideline, remarkably in terms of the key principles such as the staged TTC and usage of structure–activity relationship (SAR) evaluation. However, FDA guideline mentions

eir formation, of reduction of levels, of additional characterization of these impurities, and of considerations for flexibility in approach [22]. Along with major guidelines and supporting documents as described, there are also other documents related to impurities. Within the context of issues such as the herbal medicines, excipients of medicinal products and in ICH S9 guideline on anticancer pharmaceuticals considerable comments are also provided related to the impurity issue [23, 24, 25]. 3 Toxicological Aspects Impurities in drug substances and products can pose serious toxic effects that can be observed throughout the whole organism. One of the major targets which are affected by impurities is DNA whereby impurities generate a change in genetic material resulting in an increased overall cancer risk to human. The evaluation of identified impurities whether with mutagenic/carcinogenic effects or not as well as their control is a crucial toxicological problem which needs to be tackled. Should sufficient data that are identifying the genotoxic and carcinogenic potential, are not already available, impurities identified in drug substances or products at levels above the stated qualification thresholds should be evaluated for genotoxic potential in an initial minimal screen. In this approach it is recommended that assays be conducted with the isolated impurity. However, studies with the drug substance containing, or spiked with, the impurity may be considered, if necessary [22]. The Potency Database, CPDB (http://potency.berkeley.edu) reports the genotoxic and/or carcinogenic properties for a number of substances resulting from technical reports issued by the National Cancer Institute. Databases such as TOXNET (http://toxnet.nlm.nih.gov), NIOSH (National Institute for Occupational Safety & Health; http://www.cdc.gov.niosh), GESTIS (http://www.dguv.de/bgia/en/gestis), Discovery Gate (Symx) and PharmaPendium (Elsevier) contain information about functional groups and compounds that can react with DNA [1].


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When substance specific data on genotoxicity/carcinogenicity are not available, the impurities can be screened for the presence of structural alerts (SAs). The SAs are defined for carcinogenicity in terms of molecular functional groups or substructures that are related to the carcinogenic activity of the chemicals, thereby, chemical classes potentially able to cause cancer identified. Since many carcinogens have as a main step in there mechanism of action to attack or modify the DNA, the SAs relative to such classes of carcinogens are also valid for the mutagenicity endpoint. It should be emphasized that models based on SAs hold a special place in predictive toxicology. The current evaluation is related to the ‘‘predictive’’ power of the SAs in respect to carcinogenicity and mutagenicity. Thus, a chemical containing a SA was considered to be predicted as positive (i.e., potentially toxic), whereas a chemical without any known SA was predicted as negative [14]. A number of different lists of SAs have been reported in the literature [26- 29]. This evaluation may be conducted via a review of the available literature or through a computational toxicology assessment that are widely used of commercial (e.g., Deductive Estimation of Risk from Existing Knowledge (DEREK) https://www.lhasalimited.org/; Multi-Computer Automated Structure Evaluation (MCase) http:// www.multicase.com; Toxicity Prediction by Komputer Assisted Technology (TOPKAT) http://accelrys.com/ products/discovery-studio/predictive-toxicology.html, SciQSAR (MDL-QSAR) www.scimatics.com; Compudrug http://www.compudrug.com), and noncommercial software systems (e.g., Oncologic, by US Environmental Protection Agency http://www.epa.gov/oppt/sf/pubs/oncologic.htm). Development in computational capacity, increasing use of computer for SARs analysis medicinal chemistry as well as a fast preliminary assessment of toxicity brought forward the advancements in computational methods in toxicological assessment [1, 30]. Within the context of the pharmaceutical assessment, the potential GTIs and GTIs are identified by a careful analysis of both the degradation products and the manufacturing process. The identification of GTIs is in majority based on available toxicological data, while potential GTIs can be identified using structural alerts [1]. In the toxicological investigation of a chemical, testing a standard test battery is recommended be used by ICH and FDA guidelines related to genotoxicity testing [31- 34]. The standard test battery which includes the following was planned to avoid the risk of false negative results for compounds with genotoxic potential; i. Bacterial gene mutation test (Ames test) ii. In vitro cytogenetic evaluations of chromosomal damage in mammalian cells or in vitro mouse lymphoma thymidine kinase (TK) assay iii.Deriving on the achieved results, in vivo assessment of chromosomal damage in rodent hematopoietic cells However, the guidelines on drug impurities recommend “minimum screen” consisting of in vitro bacterial gene mutation test and in vitro chromosome aberration test for evaluation of genotoxic potential of impurities considering the international standard test battery. In addition to genotoxicity test, a repeat dose general toxicity study in a single species of 14 to 90 day duration (often a 28 day rat study) should also be conducted. Depending on the class of the impurity, additional testing may also be required. In vitro bacterial gene mutation test: The Ames mutagenicity assay is rapid and convenient shortterm bacterial reverse mutation test that is used worldwide as an initial screen to determine the mutagenic potential of compounds. For several decades, its value is recognized and it is used in research laboratories and by regulatory agencies throughout the world [35]. Within the scope of the impurities, particularly the Ames test is used as the main assay in order to confirm the genotoxic potential of an impurity highlighted as potential GTIs by a structural alert. In the Ames test, the indicator organisms for the detection of induced mutations are various strains of Salmonella typhimurium and Escherichia coli which, as a resulting of mutations in genes coding for


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enzymes for the biosynthesis of an essential amino acid (histidine or tryptophan, respectively), have lost the ability to grow on agar which lacks this amino acid. Mutagenic chemicals induce reverse mutations in the genome of these cells, i.e. the mutated cells restore the functional capability to synthesize essential histidine or tryptophan and to grow and form visible colonies in the absence of the mentioned amino acid in the agar [36]. Also, a mixture of metabolic activation, that contains liver microsomes, may be added to this test to mimic in vivo biotransformation [37]. Despite the fact that the ability of in vitro genotoxicity and mutagenicity tests to predict in vivo toxicity has limits, Ames test results have been applied as predictors for rodent carcinogenicity. It is acknowledged that the predictive power of positive Ames test results for rodent carcinogenicity is high, that is ranging from 77% to 90%. And, in the prediction of carcinogenicity no other in vitro assay is cited that better predicts carcinogenicity [38, 39, 40]. In vitro chromosome aberration test: In the testing for mutagenic/carcinogenic potential of chemicals, in vitro chromosome aberration tests have come to play a pivotal role. The aim of the in vitro chromosome aberration test is to determine agents that cause structural chromosome aberrations in cultured mammalian cells. Chromosome mutations and other relevant events are the cause of many human genetic diseases and cancer induction in humans and experimental animals causing alterations in oncogenes and tumor suppressor genes of somatic cells [41]. For the testing of a substance with regard to potential clastogenic properties, cell cultures are incubated with the test compound in the absence and the presence of an exogenous metabolic activation system. Then mitosis is arrested in metaphase with an inhibitor, due to the fact that only in this phase of the cell cycle are the chromosomes present in very compact form and amenable to an analysis of their structure by light microscopy. And the cells are fixed and prepared for chromosome analysis. A biologically significant increase in the frequency of cells with structural or numerical aberrations contrary to the control group indicates the chemical is clastogenic or aneugenic, respectively [36]. If the initial evaluation of the genotoxic potential of an impurity as described above is concluded to be negative, no further genotoxicity studies are recommended and the impurity should be considered to be sufficiently qualified regarding its genotoxic potential. Positive results, on the other hand, in one or more genotoxicity assays or other information indicating a carcinogenic potential should be addressed further [22]. In the light of the qualification results, impurities are controlled with relevant applications and approaches such as PDE, ALARP, TTC or ICHQ3 A/B limitations discussed above. Toxicology assessment is to be done by chemists and toxicologists to identify GTIs and their entry into the synthetic process, to search for the opportunities for their removal and provide limits that are consistent with safety and regulatory expectations [15]. It is observed that general focus in the worldwide literature database is on analysis orientated identification, quantification and characterization of impurities in drugs and discussion on analytical methods in research on drug impurities. However, there are limited scientific literatures on investigating toxicities of the impurities within the scope of toxicology. Agarwal et al’s study which evaluates the acute toxicity and genotoxicity data of the dimeric impurity of cefotaxime, is one example of the recent studies investigating toxicity of impurities in the literature. In this study, Ames, chromosome aberration and acute toxicity tests are conducted and indicated that the dimeric impurity of cefotaxime is nonmutagenic in Ames test, nonclastogenic in vitro, and acutely nontoxic in rats [42]. Other study, conducted by Eichenbaum et.al, aimed to completely characterize the genotoxic potential of p-nitrophenol (PNP) existing as a drug impurity. Aiming at completing the gaps in existing study results, in vivo mouse micronucleus and dermal pharmacokinetic bridging studies were conducted. Researchers indicated that PNP should be considered a non-genotoxic impurity and, as a drug impurity, a threshold limit of  4 mg/ day would be set in accordance with ICHQ3C [43]. The study for providing information to establish the safety profile of the piperacillin impurity-A indicated non-mutagenic in Ames


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test and non-clastogenic in chromosome aberration test of the impurity [44]. Genotoxicity of levofloxacin noxide which is an impurity isolated from levofloxacin was investigated by DEREK software system for in silico assessment and in vitro methods as mouse lymphoma assay and in vitro chromosome aberration assay. According to these assays, the impurity might be controlled as a nongenotoxic impurity despite of the DEREK alerting [45]. Toxicological considerations on impurity exposures for pediatric and other sensitive population Children have clearly much different exposure patterns than adults. By deduction chemicals at different levels affect children differently than adults due to differences in behavior, in physiology and physical differences. These influence the risk assessment associated with exposure to chemicals. It should be noted that children have more years of life ahead of them than adults, so they have longer periods to develop chronic diseases from exposure to chemicals [46]. From the aspect of impurities, particularly in the use of chronic drugs exposure to impurities also become chronic. This especially in the existence of GTIs may cause many unwanted consequences. Depending on stage of maturation or unique susceptibilities populations face carcinogenic risks differently. Although there is not much data to characterize variable risks to different populations, there is support for young individuals being more susceptible to carcinogenicity than older individuals [17]. Thus, the sensitive subpopulations should always be handled as an important consideration for toxicological evaluations. And FDA guidance for GTIs recommends that different patient populations, particularly pediatric populations, need to have lower thresholds because of the higher cancer susceptibility in pediatrics. In the EPA guidance Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens (EPA/630/R-03/003F), the adjustment is applied as the additional safety factors of 10 for exposures before 2 years of age while the safety factor of 3 is used for exposures between 2 and less than 16 years of age. If other sensitive subpopulations of individuals are identified, these predispositions can be factored into the allowable daily intake. 4 Analytical Perspectives In order to determine biological safety, isolation and characterization of impurities is required for acquiring and evaluating data, thereby revealing the need and scope of impurity profiling of drugs in pharmaceutical research [7]. According to guidelines, the documented evidence that the analytical procedures have been validated and are suitable for the detection and quantitation of impurities should be included in the registration applications [6, 8]. Of all the impurities, the profiling of organic impurities can be more challenging. Also, the analysis of GTIs in drug product can be more challenging than drug substance, simply because of the matrix issue and much lower sensitivity limit needed while working with drug product [47]. To isolate and quantify the impurities, various instrumental analytical techniques are routinely been employed. The entire analytical procedure can be devoted into three operations: sample preparation, separation and detection. Due to the fact that since the choice of the method for sample preparation can dictate the corresponding separation and detection techniques, it should be acknowledged that each operation is potentially interconnected with the others. Therefore, the three parts should be evaluated as a whole [1]. Selection of appropriate technique depends upon the nature of sample to be analyzed (i.e. its structure, physicochemical properties, availability) and types and stability of impurities present. Tools for impurity analysis mainly include spectroscopic and chromatographic methods or a combination of both [48]. Spectroscopy is one of the most powerful tools which are helpful for the study of atomic and molecular structure and there are several spectroscopic techniques such as Ultraviolet Spectroscopy (UV), Fourier Transform Infra Red Spectroscopy (FTIR), Nuclear Magnetic Resonance Spectroscopy (NMR), Mass


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Spectroscopy (MS), which are used for the characterization of impurities and degradants. Chromatographic Techniques such as Thin Layer Chromatography (TLC), High Performance Thin Layer Chromatography (HPTLC), High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), Capillary Electrophoresis (CE); are regularly utilized for the identification and quantification of an impurity of pharmaceuticals. In addition, hyphenated techniques such as LC-MS, GC-MS, LC-NMR, CE-MS and Inductively Coupled Plasma Mass Spectrometry (ICP-MS) have been developed to solve various complex analytical problems in pharmaceutical industry. One of the advantages for hyphenated techniques is the sample requirement which is less compared to conventional techniques [48]. There are plenty of papers which have discussed the present state of the art level of analytical techniques for the determination of impurities of important drug groups [5, 49- 55]. In the determination of the sensitivity requirements for the analytical method, the specification limit for an impurity is the key factor and it can often dictate the appropriate technique for detection. At the level of 100 ppm, LC with UV detection and GC with flame ionization detection are often adequate. In the range of 10–1 ppm or lower, even though in some cases UV detection can still be applied with success, hyphenated MS techniques such as LC–MS and GC–MS are by far the most appropriate techniques [1]. These techniques, due to sensitivity and selectivity, have been widely used in GTI analysis. Although GC is a very well known technique for organic volatile impurities, it is reported that HPLC has been the main technique used for analysis of impurities in drugs compared to other techniques [49]. Most of the users have conducted the reversed-phase mode with UV absorbance detection because of the best available reliability, analysis time, repeatability and sensitivity. However, in some cases, more novel approaches are used that are often based on separation mode or the use of more selective detective systems typically diode array and fluorescence detectors or mass spectrometers. Residual solvents are typically determined using chromatographic techniques such as gas chromatography. In the determination of levels of residual solvents as described in the pharmacopoeias, harmonized procedures should be used, if possible. Otherwise, manufacturers would be free to select the most appropriate validated analytical procedure for a particular application [9, 10]. Many procedures have been developed for selective detection and quantification of metal species. In some procedures, it is a fact that excitation and emission phenomena are used to detect metals in intact material, such as X-ray fluorescence and neutron activation analysis. Other procedures which require an initial atomization and ionization process separate the metals from the organic matrix. The process includes using flame, furnace, plasma, laser, or spark techniques. After ionization, the metals are quantified using optical emission, chromatographic techniques, or mass spectrometry. Due to restraints in methods of sufficient sensitivity and selectivity for toxicologically based metal limits,the analytical methodologies applied to most inorganic impurities as in ICP Optical Emission Spectroscopy (ICP-OES) and ICP-MS, Atomic Absorption Spectrometer (AAS), and Atomic Emission Spectroscopy (AES) are the most suitable procedures [13]. There are many published analytical methods that describe the analysis of impurities in pharmaceuticals. The available literature on impurity analysis is continuously increasing. 5 Conclusion Impurities can have serious toxicological effects, the existence of these impurities and their levels in pharmaceuticals could have crucial impacts on the quality of drug products and the safety of patients. Ideally drugs are expected to be free from impurities, but this is often unattainable in practice because of various reasons. On the other hand, with further improved technological capability in analyzing impurities accompanied by growing concern about their potential toxicity on human health the need to control the


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impurities in pharmaceuticals via regulations has become even more evident. The existing guidelines published by regulatory authorities provide information about impurities within a range from identification to controlling which covers toxicological risk assessment as well as analytical approaches. In addition, GTIs especially bring along significant concern in the aspect of risk/benefit and together with studies that continue on with regard to these issues the debate on them also persists. Some of the approaches which are currently used for controlling impurities could be referred as the TTC concept as a pragmatic solution, PDE restricting and the ALARP principle. In order to pursue complementary assessments for impurities there is a clear need for a multi-disciplinary collaboration of experts in the field of toxicology, synthetic and analytical chemistry and others if required. Raising awareness of health authorities, pharmaceutical industry, academic community and governments with all their relevant bodies and institutions followed by efforts which are in line with this and regulatory mechanisms will be important steps in protecting public health. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

13. 14. 15. 16. 17.

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