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  • Assessment of Genotoxic Impurities

    In Pharmaceuticals, the manufacturers of APIs and Drug products have the findings of impurities that provide no therapeutic benefits but have potential harm which may be likely because the human carcinogens in the drugs were of great concern. Genotoxicity is the property of chemical agents that damage the genetic information within a cell causing mutation which may show and lead to cancer. The main source of genotoxic impurities is the starting raw materials used for the manufacturing of drug substances, solvents, reagents, and catalysts.

    International Conference on Harmonization (ICH) Q3A Impurities in New Drug Substance and Q3B (R2) Impurities in New Drug Products provides guidance for the qualification and control for most of the impurities, M7 (R2) for assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk on DNA-reactive substances that could potentially cause damage when present at low levels and potentially cause cancer.

    A potential genotoxic impurity (PGI) has been defined as an “Impurity that shows a structural alert for genotoxicity but that has not been tested in an experimental test model. Here potentially relates to genotoxicity, not to the presence or absence of this impurity”. Genotoxic impurities impact the genetic material using mutations through chromosomal breaks, rearrangements, covalent binding, or insertion into the DNA during replication which may result in carcinogenesis.

    The guidance document features sections on considerations for marketed products, drug substance, and drug product impurity assessments, hazard assessment elements, risk characterization, control, documentation, and three appendices on scenarios for the application of ICH M7, case examples to illustrate potential control approaches and an addendum to M7.

    Sources of Genotoxic Impurities

    Genotoxic impurities (GTIs) are expected to get into drug substances through several sources, the main source is starting material (the raw material used for the manufacture of drug substance), and its impurities, also genotoxic intermediate and by-products formed in the synthesis process may get to be carried forward to the drug substances as genotoxic impurities. In addition to these, solvents, catalysts, and reagents used in the synthesis process can also be probable sources of genotoxic impurities in drug substances.

    Degradation products generated on storage and shipment or exposure to light, air oxidation, or hydrolysis contribute to the generation of impurities in drug substances. Besides these excipients and their impurities, extractable and leachable can also contribute to genotoxic impurities in drug products, following is the representation in Table:1 (Genotoxic compounds in drug substances)

    Category/StageCompounds
    Starting materialHydrazine, Nitroso, and acrylonitrile compounds
    IntermediateBenzaldehyde, Nitro compounds
    By-productSulphonate esters, phosgene
    ReagentFormaldehyde, epoxides, esters of phosphate & sulphonates
    SolventBenzene, 1,2-dichloroethane
    CatalystToxic heavy metals, metal phosphates
    Degradation productN-oxides, aldehydes,
    Table 1: Genotoxic compounds in drug substances

    Some examples of alerting functional groups that are known to be involved in reactions with DNA. These functional groups were categorized into three groups Aromatic, Alkyl, and Aryl & Heteroatomic groups as given below.

    Industrial approach: The Pharmaceutical Research Manufactures Association (PhRMA) group has proposed a model in which all impurities in drug substances be classified into five classes:

    CLASSDefinition
    Class 1Impurities known to be genotoxic (mutagenic) and carcinogenic
    Class 2Impurities known to be genotoxic (mutagenic) but with unknown carcinogenic potential
    Class 3Impurities containing alerting structures, unrelated to the structure of the API and of unknown genotoxic (mutagenic) potential
    Class 4Impurities containing alerting structures which are related to the API
    Class 5Impurities with no alerting structures or where sufficient evidence exists that genotoxicity is absent

    Control of selected Genotoxic Impurities in APIs:

    GTIs can be well controlled by the process, without affecting quality, by modifying the route of synthesis. Methods for controlling sulfonates and alkylating agents.

    Limits for Impurities

    Guidelines from the ICH and EMA provide the limits for impurities in drug substances and drug products. These limits do not apply to GTIs because of their adverse effects; hence it is necessary to determine limits based on the daily dose of the drug substance. This task drains process-development resources. To overcome this problem, scientists have to identify GTIs early in process development, develop analytical methods (i.e., for quantifying the genotoxic impurity), and demonstrate the necessary synthetic process controls.

    Allowable DurationThe threshold of Toxicological Concern (TTC) limits corresponding
    to the duration of dosing,
    USFDA (Control Threshold (µg/Day)EMA (Control Threshold (µg/Day)
    ≤ 1 month120120
    ≥ 1-12 months2060
    ≥ 1-10 years1030
    > 10 years to Lifetime1.55
    Table 2: TTC and LTL (less than lifetime limits) safety-based limits for mutagenic impurities

    Different Classes of Mutagenic Impurities with Control Strategies

    In silico, structure-based assessments, i.e. Derek Nexus, Sarah Nexus, etc., are used for predicting mutagenicity based on QSAR (quantitative structure-activity relationships) approaches. These findings are then reviewed by toxicology experts to provide any additional understanding as to the relevance of these predictions (both positive and negative), and in the case of contradictory outcomes to understand those differences. Based on this assessment, impurities are categorized into 5 different classes in order of decreasing regulatory concern.

    Impurity ClassCommentaryControl Strategy
    1Known mutagenic carcinogensControl at or below compound’s specific acceptable limit, i.e. AIs or PDEs1
    2Known mutagens with unknown carcinogenic potentialControl at or below acceptable limits, i.e. LTL2 or TTC 3
    3Show alerting structures (un-related to drug substance) with no supporting mutagenicity dataControl at or below acceptable limits, i.e. LTL2 or TTC3, Or conduct bacterial mutagenicity assay; If non-mutagenic = Class 5 If mutagenic = Class 2
    4Show alerting structures (related to drug substance which is itself nonmutagenic)Treat as a non-mutagenic impurity, i.e. use default ICH Q3A/Q3B limits
    5Show no alerting structuresTreat as a non-mutagenic impurity, i.e. use default ICH Q3A/Q3B limits
    Table 3: Different Classes of Potential or Real Mutagenic Impurities Based on Mutagenic and Carcinogenic Potential and Proposed Control Strategies.

    Analytical Challenges

    Analysis of genotoxic impurities can be very challenging because they must be controlled at levels significantly lower in the range of 1 to 5 ppm. Such low levels require more sensitive analytical instruments. Depending on the nature and quantity of the genotoxic impurity being investigated, an appropriate analytical technique needs to be selected.

    The identification and control of potential genotoxic impurities in a synthetic process are always challenging, due to their growing nature and mutable points of entry. Therefore, synthetic routes must be screened for the identification of structural alerts, which causes genotoxicity. Genotoxic impurity profiling in API & Pharmaceutical compounds plays a vital role in clinical development. Hence, a specific, accurate, and robust analytical method needs to be employed for the detection and control of these genotoxic impurities below the TTC level.

    Hyphenated technique for better separation

    Figure 1. Lower detection levels of genotoxic impurities require more sophisticated analytical techniques for quantification.

    The identification and control of potential genotoxic impurities in a synthetic process are always challenging, due to their growing nature and mutable points of entry. Therefore, synthetic routes must be screened for the identification of structural alerts, which causes genotoxicity. Genotoxic impurity profiling in API & Pharmaceutical compounds plays a vital role in clinical development. Hence, a specific, accurate, and robust analytical method needs to be employed for the detection and control of these genotoxic impurities below the TTC level.

    FAQ

    What are the sources of genotoxic impurities?

    Degradation products generated on storage and shipment or exposure to light, air oxidation or hydrolysis contribute to the generation of impurities in drug substances. Besides these excipients and their impurities, extractable and leachable can also contribute to genotoxic impurities in drug products.

    What are the effects of genotoxic impurities?

    Genotoxicity is the property of chemical agents that damage the genetic information within a cell causing mutation which may show and lead to cancer. The main source of genotoxic impurities is the starting raw materials used for the manufacturing of drug substances, solvents, reagents, and catalysts.

    What are Class 1 genotoxic impurities?

    Class 1 Impurities are known to be genotoxic (mutagenic) and carcinogenic and known to be at risk and need to eliminate by modifying the process.

    References:

    1. International Conference on Harmonization, ICH M7 (R1): Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk
    2. International Conference on Harmonization, ICH Q3B (R2): Impurities in New Drug Products: ICH.
    3. International Conference on Harmonization, ICH Q3C (R4): Impurities: Residual Solvents: ICH.
    4. Kruhlak, NL, et al. Progress in QSAR toxicity screening of pharmaceutical impurities and other FDA-regulated products. Adv Drug Deliv Rev. 2007;59:43-55.
    5. European Medicines Agency, Guideline on the Limits of Genotoxic Impurities, EMA/CHMP/ICH/83812/2013
    6. Liu DQ and Korda AS. Analytical challenge instability testing for genotoxic impurities. Trends Anal Chem. 2013;49:108-117.
    7. Baker A. Development of a strategy for analysis of genotoxic impurities. Genotoxic impurities, strategies for identification and control. Wiley Publication. 2011:281-304.
    8. Snodin, DJ. Genotoxic impurities: From structural alerts to qualification. Org Process Res Dev. 2010;14:960-976.
    9. Robinson Derek I (2010) Control of genotoxic impurities in active pharmaceutical ingredients: a review and perspective. Organic Process Research & Development 14(4): 946-959.

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