There was no specific document on control of genotoxic impurities before 2000. ICH guidelines made passing references to
compounds of unusual toxicity. Genotoxic impurities are chemical compounds that may be mutagenic and could potentially damage
DNA (76). Non-monoalkylated agents are classified as genotoxic due to the nature of the functional groups they possess and
also of related aniline derivatives. Additionally, salt-forming steps can introduce genotoxic impurities. Some examples include
formation of methyl chloride as a side reaction of hydrochloric acid in methanol or esters of methanesulfonic acid as byproducts
from the methanesulfonic acid salt-formation step in alcohol-based solvents (77, 78).
EMA issued guidelines on the threshold of toxicological concern (TTC) that recommended limits for exposure to potential genotoxic
impurities to be 1.5 mcg per day for commercially approved drugs (79). As per the guidelines, testing will be required for
all potential impurities from an API's synthetic route containing structural elements that are the cause of concern for genotoxicity
potential using the well-established Salmonelle Ames test. The Ames test is a screening test that is used to help identify
chemicals that affect the structure of DNA. The test exposes Salmonella bacteria to chemicals and looks for changes in the
way bacteria grow. These changes result from mutations that occur when the structure of DNA is altered in certain places and
the micronuclei test for mutagenicity (80, 81). Recommended qualification thresholds based on the maximum daily dose for drug
substances and for drug products are provided in ICH Q3A and ICH Q3b (7, 8). The TTC data set was conducted from the perspective
of an organic chemist who develops process technology for APIs (82). As part of the EMA guidance, API process designers are
instructed to avoid all possible situations that could lead to the presence of impurities possessing genotoxic potential at
any level in APIs.
During the establishing of the control mechanism, other factors, such as reactivity, solubility, and volatility, should be
considered. Action should not be based only on the presence of alerting structures. It is important to make evaluations on
a case-by-case basis, and precedence data should be considered, such as the stage of impurity formation, reactivity and carryover
to the API, the intake of other routes, Ames test results, and data of closely related structures.
During process development, a genotoxic impurity may be introduced as a starting material, reagent, intermediate, catalyst,
byproduct, isomer, or degradation product. (83). Alkyl halides used as reagents in synthesis are genotoxins (84). The same
also was generated during chemical synthesis when a salt counter ion (e.g., hydrogen halide) of a drug substance reacts with
alcohols when used as a solvent media.
The genotoxins ethyl chloride, methyl chloride, and isopropyl chloride were generated during the preparation of the hydrochloride
salts of ethanol, methanol, and isopropyl alcohol (ICH listed solvents), respectively, at lower temperature (< 5 °C) as the
key parameter of these impurities. In alcohol solvents, when HCl was 37% aqueous HCl or gas, it creates the maximum chance
to form these alkyl halide impurities at trace levels. These impurities are detectable in GC at ppm level. Methane sulfonic
acid (mesylate), benzene sulfonic acid (besylate) and p-toluenesulfonic acid (tosylate) are commonly used as counter ions to form API salts (85–87). Interactions of these acids
with residual alcohols may lead to the generation of genotoxic impurities. Alkyl methane sulfonates, alkyl benzene sulfonates,
and alkyl para-toluene sulfonates may combine with imatinib mesylate, amlodipine besylate, and denagliptin tosylate, respectively
The emphasis on genotoxic impurities is increasing, which creates challenges for both synthetic and analytical chemists, to
develop sensitive and efficient methods to detect impurities at low levels (i.e., below TTC < 1.5 mcg/per day), which sometimes
is not feasible and which increases the time and cost of drug development.
Linezolid (S)-N-[[3-[3-fluoro-4-(4-morpholinyl)phenyl]-2-oxo-5-oxazolidinyl]methyl]-acetamide, has genotoxic structural alerts and represents
a new class of antibiotics, oxazolidinones. Forced-degradation studies are an important part of the drug-development process
and are used increasingly in testing new molecules. These studies may give different impurities that may not be formed during
process optimization and manufacturing validation, but these impurities must be controlled as per ICH guidelines. The authors
have observed two impurities during a forced-degradation study in peroxide and alkaline conditions, Compounds 7, 8, and 9
(see Figure 4), which are structural alerts for genotoxicity, and which should be controlled so that the exposure to it is
less than 1.5 mcg/day based on the maximum daily dose of the linezolid.
Figure 4: Process, genotoxic, and metabolite impurities of linezolid.
Linezolid's key starting material (A) shows genotoxicity alert and it contains five other intermediates, Compounds 1, 2, 3,
4, and 5 (see Figure 4). Compound A converts to the final drug, and it contains, Mesyl Impurity 6, Amine Impurity 12, Des
Fluoro Impurity 13, Chloro Impurity 14, and O-Acetyl Impurity 15; these are the process impurities and have genotoxicity alert
(49). During human studies, from the total amount of linezolid administered, only 30% was eliminated through the kidneys.
Its major part was metabolized by oxidation of its morpholine ring, which resulted in the formation of two metabolites (see
Figure 4): amino ethoxy acetic acid metabolite and hydroxy ethyl glycine metabolite (i.e., a major urinary metabolite) (90–92).
Part II of this article examined impurities that are associated with drug molecules having one or more chiral centers, APIs
existing in various crystalline forms, drug substances with the piepererazine moiety, and APIs developed by new processes.
Part II also looked into the extended application of the TTC to pharmaceuticals. To guarantee the quality and safety of pharmaceuticals
during drug development, a quality concept has been proposed that adapts the ICH guidelines and which is focused on qualified
Call for Papers
Part III, to be published in the April 2012 issue of Pharmaceutical Technology, will examine various degradation routes of APIs, impurities arising from API–excipient interaction during formulation, metabolite
impurities, various analytical methods to measure impurity levels, and measures to control impurities. Part I, published in
the February issue of Pharmaceutical Technology, examined the types and sources of impurities (11).
Kashyap R. Wadekar, PhD,* is a research scientist (II), Mitali Bhalme, PhD, is an associate research scientist, S. Srinivasa Rao is a research associate, K. Vigneshwar Reddy is a research associate, L. Sampath Kumar is a research chemist, E. Balasubrahmanyam is a research chemist, and Ponnaiah Ravi, PhD, is senior vice-president of R&D, all with Neuland Laboratories, 204 Meridian Plaza, 6-3-854/1, Ameerpet, Hyderabad, India,
tel. 91 40 30211600, firstname.lastname@example.org