Assessment of Genotoxic Impurities

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 (GTIs) 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 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

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

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 DurationThreshold 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 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 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.

ICH Identification LimitGenotoxic Impurities Typical Limits 
0.1 % to 1000 ppm0.01 % to 100 ppm0.001 % to 10 ppm0.0001 % 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 the detection and control of these genotoxic impurities below the TTC level.


  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.

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