Why is it Difficult to Detect Mutagenic Impurities?

The guidelines for detecting pharmaceutical impurities, including ICH M7, which are either known or suspected to be mutagenic are meant to give a broad picture of the limits to which these may be present. Such impurities occur in association with known additives, or come from the environmental influence or by a process of breakdown of a drug.

The limits for such impurities, as now proposed, lie much below the level of the common impurities that the ICH Q3A deals with and their detection needs more sensitive analytical techniques, which can pick up levels from ppm to ppb. This article describes the general analytic techniques for this purpose.


Safety and effectiveness are paramount in the development of an investigational new drug. Safety limits are becoming clear in the case of CMC over the past 20 years, in particular, the assessment of impurities in active drugs and drug products, when the container is closed and during the manufacturing process, is discussed in several guidelines such as the ICH, the USP and by agencies that regulate drugs.

Newer guidelines have emerged for the evaluation of trace metals and mutagenic impurities, which indicate the will to control impurities strongly. The ICH M7 guidelines are meant to indicate ways to restrict the risk of carcinogenicity as it evaluates the presence of possible mutagens in new drug materials or drug products. The fundamental difficulty is the very low concentration of the mutagenic impurities (MI) at near limit-of-detection levels.

In the early stages, articles and draft guidelines dealt with genotoxic and carcinogenic impurities, these being the terms used for what are now termed mutagenic impurities since the M7 guidelines were issued in 2015. This is significant because genotoxins are not always mutagens, which are defined as below:

“Anything that causes a mutation (a change in the DNA of a cell). DNA changes caused by mutagens may harm cells and cause certain diseases, such as cancer. Examples of mutagens include radioactive substances, x-rays, ultraviolet radiation, and certain chemical.”

Quantifying MIs

Impurities other than MIs are generally measured in drug substances at concentrations exceeding 0.05% weight/weight or as the relative peak area, and the techniques used are standard according to ICH Q3A. The critical levels of MIs are set by the daily drug intake and the duration of dosage, and when the concentration is less than 10 ppm they are below 1.5 µg per day.

This means that techniques which can detect concentrations 70 times less than standard are needed, as given in Table 1. One approach is to classify MI entry from the three main sources, as the difficulty of detection varies with the source.

Table 1. Comparing Q3A and M7 levels.

Comparing Q3A and M7 levels.

As the table shows, MI measurement needs techniques with much higher sensitivity than would be required for other impurities by the Q3A standard at a concentration of 0.05% and at a 30% threshold of toxicological concern (TTC).

This article describes two primary sources of MI, namely, added substances and those which form within the matrix. There is a third source, called environmental MIs or leachable MIs which are analyzed in other types of programs and are not discussed here.

Added MIs

Following an initial evaluation, the workflow for detection is laid out. In the case of added MIs the detection is simpler. For instance, if acid chloride addition at step 3 of five steps in a synthesis is known to have happened, a sample can be used with extrapolation of the characteristics of detection, using the toxicological data on hand to make the evaluation simpler.

When an added MI requires testing of the final drug substance or the intermediate, the assessment also includes evaluating a technique for separation and another for detection. The following factors must be considered:

  • Whether the current method of analysis detects the MI and at what limit
  • The required detection or quantification limit as per TTC
  • Whether the compound is volatile
  • The ionization characteristic of the MI as per expectation, and whether it is suitable for MS
  • The reactivity of the MI and the need for derivatization

Added MIs are typically more reactive and any method developed should therefore account for this reactivity so that accuracy can be maintained even after spiking API into samples, by ensuring that these MIs remain stable. Alkyl halides are MIs which react with amines, and this reduces accuracy of detection in GC headspace tests for recovery studies.

MIs Formed

MIs are formed from degradation of drug substances or from the matrix or by the process. Their detection is then more complex. If the degradation product has toxicity as defined by Q3A(R2) standards, as shown in Figure 1, more care must be taken as this set of standards has a gap in the decision tree and the note on it, summarized in the statement: “Lower thresholds can be appropriate if the degradation product is unusually toxic.”

This addresses toxic degradation products but at the same time does not suggest the need for identification. A look at the decision tree suggests that the latter should be reduced to subthreshold levels rather than requiring further action. The note however seems to take it for granted that the product is first identified by mentioning unusual toxicity. Thus M7 offers better authority for MI assessment than Q3A R2.

A Q3B(R2) decision tree for the identification and qualification of a degradation product

Figure 1. A Q3B(R2) decision tree for the identification and qualification of a degradation product

Taking one instance of a worst-case scenario in which the assessment following M7 standards finds what appears to be a degradation product of concern in API, or two actives and a number of excipients are detected in the corresponding drug product, it may be followed up by deliberately subjecting the drug substance to stress in order to detect the product of concern, followed by in-silico analysis and bacterial assay. In addition, some points must be considered:

  • Should the degradation product be isolated or synthesized to confirm its structure, obtain material for analytical reference, and for in vivo testing?
  • Should it be monitored in the complex drug product during long-term stability studies?

In one case, if an in-silico MI of concern is found as a functional group forming part of the primary structure, as for example, a substituted aniline, then any degradation product, whether known or only proposed, which contains this functional group, will set off the in silico alert. While the general principle is that if the parent molecule is non-mutagenic, degradation products of the same type will be non-mutagenic as well, but conducting a risk assessment is advised. As per ICH suggestions, M7 should not be applied when advanced cancer drugs are being studied.

Techniques of Detection

Quantitative assessment of impurities at low concentrations is more easily done by some techniques than by others. Table 2 shows listed detectors according to their general sensitivity, in which UV has been assigned a value of 1. According to this arbitrary scale, an electrochemical detector has a ten-fold greater sensitivity than UV. The general sensitivities depend greatly on the specific compound.

Table 2. General Sensitivity Overview-HPLC Detectors

General Sensitivity Overview-HPLC Detectors

Mass spectrometry is the most sensitive detection method, with the additional capability to identify the compound. For instance, using a trap MS which can monitor single ions, along with tools such as a Q Executive® Orbitrap results in quantitative measurement of low levels of MIs in a complex matrix, which makes it of great value when MIs are to be screened for or monitored, or both.

Table 3. GC Detector Sensitivity4

GC Detector Sensitivity

Thus MI detection and quantitation requires the use of knowledge gained from multiple fields, including synthesis, toxicology, analysis and manufacturing, to evolve the right mix of techniques to detect and measure these compounds, while continuing to test the drug product throughout the process of drug development.


  • MIs arise from three primary sources:
    • Added MIs including impurities coming from the process
    • Environmental contaminants
    • Degradation products
  • MI characterization often needs the capability to detect compounds at low levels
  • Multiple options are available for detection, and many tools
  • Advanced MS is a valuable option for identification and quantitative assessment of MIs
  • MI identification and quantitation varies in difficulty level, based on whether it is already known, the properties of the compound, and the threshold of detection that is required


  1. M7(R1) Addendum to ICH M7: Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk. Retrieved from https://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM464285.pdfRetrieved from, https://www.cancer.gov/publications/dictionaries/cancer-terms?cdrid=601170
  2. ICH Q3A R2, Guidance Impurities in New Drug Substances
  3. NCI Dictionary of Terms, Mutagen. Retrieved from https://www.cancer.gov/publications/dictionaries/cancer-terms?cdrid=601170
  4. Hutchinson J. et al., (2011). Investigating universal detection systems for the analysis of pharmaceutical preparation. Retrieved from http://www.cosmoscience.org/archives/2011/Investigating%20Universal%20Detection%20System%20_Hutchinson.pdf

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Last updated: Aug 22, 2018 at 8:17 AM

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