Starting materials, reagents, intermediates, byproducts, and degraded products are often found as impurities in active pharmaceutical
ingredients (APIs). Some of these known impurities are mutagens or carcinogens with the potential to cause adverse effects
on the human body, even at trace levels. Control and determination of these impurities at parts-per-million or parts-per-billion
levels are significant challenges for analysts. When developing synthetic routes to APIs, it is the primary responsibility
of laboratory personnel to identify the stages in which impurity generation can occur. The analyst must also identify and
determine genotoxic impurities (GTIs) and control them at the stages of formation (see Figure 1). The toxicologist must perform
safety evaluations of high-priority compounds, known API impurities, and impurities with a high probability of occurring,
and subsequently classify these compounds as genotoxic or routine impurities and propose limits. Various chromatography and
spectroscopy methods help identify GTIs in an API. To detect and quantify the required levels and determine the signal-to-noise
ratio, a derivatization must be performed, provided interference does not occur. This article will describe how to select
methods for determining and controlling GTIs, based on their properties.
Figure 1: Representative structures of potential genotoxic impurities. (ALL FIGURES ARE COURTESY OF THE AUTHORS)
Limits for impurities
Guidelines from the International Conference on Harmonization (ICH) and EMA provide the limits for impurities in drug substances
and drug products (1–3). These limits do not apply to GTIs because of their adverse affects, 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.
EMA guidelines classify GTIs into two categories. The first, GTIs with sufficient experimental evidence for a threshold-related
mechanism, is regulated using methods outlined in ICH Q3C (R4) for Class 2 solvents (4). For the second category, GTIs without
sufficient experimental evidence for a threshold-related mechanism, EMA proposes a "threshold of toxicological concern (TTC)."
A TTC value of 1.5 g/day intake of a GTI is considered to be associated with an acceptable risk (see Table I) (5). The concentration
limit in ppm of GTI permitted in a drug substance is the ratio of TTC in μg/day and daily dose in g/day. EMA also released
a "Question and Answer" document to clarify questions that arose from its original guidance (6, 7).
Table I: FDA and EMA recommend acceptable qualification thresholds for genotoxic impurities in pharmaceuticals used in clinical
The Pharmaceutical Research and Manufacturers Association's approach.
The Pharmaceutical Research and Manufacturers Association (PhRMA) published a procedure for the testing, classification, qualification,
and toxicological risk assessment of GTIs (8). It listed functional groups known to be involved in reactions with DNA that
could be used as structural alerts. These functional groups were categorized into aromatic groups (e.g., N-hydroxyaryls, N-acylated aminoaryls, aza-aryl N-oxides, aminoaryls, and alkylated aminoaryls), alkyl and aryl groups (e.g., aldehydes, N-methylols, N-nitrosamines, nitro compounds, carbamates, epoxides, aziridines, propiolactones, propiosultones, N or S mustards, hydrazines, and azo compounds), and hetero aromatic groups (e.g., Michael-reactive acceptors, alkylesters of phosphonates
or sulfonates, haloalkenes, and primary halides).
PhRMA also categorized impurities into five classes. Class 1 impurities are genotoxic (i.e., mutagenic) and carcinogenic.
These impurities represent the most serious risk, and the default preference is to eliminate them by modifying the process.
If this is not possible, the TTC limit can be employed as a last resort. Class 2 impurities are genotoxic, but their carcinogenic
potential is not known. These impurities are to be controlled using TTC principles. Class 3 impurities contain problematic
structures unrelated to the structure of the API and of unknown genotoxic potential. This group includes impurities with functional
moieties that can be linked to genotoxicity based on structure. Class 4 impurities contain problematic structures related
to the API. These impurities contain a potentially worrisome functional moiety shared with the parent structure. Class 5 impurities
have no problematic structures, and evidence indicates the absence of genotoxicity. These compounds are to be treated as normal
impurities and controlled according to the ICH guidelines. If Class 3 or 4 compounds are genotoxic or not tested, they are
moved into class 2. If these are nongenotoxic, they are considered as Class 5.