The US Food and Drug Administration (FDA) finalized International Conference on Harmonization (ICH) guidance 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 (GTI’s) are expected to get into drug substances through several sources, the main source is starting material and its impurities. Also, genotoxic intermediate and by-products formed in the synthesis process may get 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 a probable source 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. Beside these excipients and their impurities, extractable and leachable can also contribute to genotoxic impurities in drug products, following is the representation in Table 1
|Starting material||Hydrazine, Nitroso, acrylonitrile compounds|
|Intermediate||Benzaldehyde, Nitro compounds|
|By-product||Sulphonate esters, phosgene|
|Reagent||Formaldehyde, epoxides, esters of phosphate & sulphonates|
|Catalyst||Toxic heavy metals, metal phosphates|
|Degradation product||N-oxides, aldehydes,|
Table 1: Genotoxic compounds in drug substances
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 Duration||Threshold of Toxicological Concern (TTC) limits corresponding to the duration of dosing,|
|USFDA (Control Threshold (µg/Day)||EMA (Control Threshold (µg/Day)|
|≤ 1 month||120||120|
|≥ 1-12 months||20||60|
|≥ 1-10 years||10||30|
|> 10 years to Lifetime||1.5||5|
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 upon 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 Class||Commentary||Control Strategy|
|1||Known mutagenic carcinogens||Control at or below compounds specific acceptable limit, i.e. AIs or PDEs1|
|2||Known mutagens with unknown carcinogenic potential||Control at or below acceptable limits, i.e. LTL2 or TTC 3|
|3||Show alerting structures (un-related to drug substance) with no supporting mutagenicity data||Control at or below acceptable limits, i.e. LTL2 or TTC3, Or conduct bacterial mutagenicity assay; If non-mutagenic = Class 5 If mutagenic = Class 2|
|4||Show alerting structures (related to drug substance which is itself nonmutagenic)||Treat as a non-mutagenic impurity, i.e. use default ICH Q3A/Q3B limits|
|5||Show no alerting structures||Treat as 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.
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.
|ICH Identification Limit||Genotoxic Impurities Typical Limits|
|0.1 % to 1000 ppm||0.01 % to 100 ppm||0.001 % to 10 ppm||0.0001 % to 1 ppm|
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 detection and control of these genotoxic impurities below TTC level.