Control of Genotoxic Impurities as a Critical Quality Attribute

For the past few years identification and control of genotoxic impurities in pharmaceutical products has become worldwide one of key issues within development and commercialization of the products. Since some of these impurities may cause mutations and potentially a cancer, there are efforts to avoid and/or keep them to minimal levels to limit the potential carcinogenic risks and so ensure the safety of the products.

Consequently, both drug developers and drug producers including API suppliers are investing vast resources in assessments of potential impurities in their products and in developing analytical methods to determine and control the toxic ones to assure compliance of their products with the relevant health regulations.

Genotoxic impurities in pharmaceutical products

Impurities in pharmaceutical products result from their synthesis and/or subsequent degradation and so their occurrence of the impurities cannot be completely avoided. They are defined as substances that yield no therapeutic benefit, but have the potential to cause adverse effects. Thus, impurity levels in the products must be controlled to ensure their safety. Some of the impurities are genotoxic and may cause deleterious changes in the genetic material of cells. The genotoxic compounds, which have a potential to directly damage DNA through chemical reactions with DNA, even if present at very low levels, leading to mutations and also subsequently potentially cause a cancer, are denominated as DNA-reactive or mutagenic. They may also act as mutagenic carcinogens. Thus, they should be controlled at levels  expected to pose negligible carcinogenic risks. These levels are much lower than those for ordinary impurities. Other types of genotoxic compounds that are non-mutagenic (e.g. clastogens) typically have threshold mechanisms and usually do not pose any carcinogenic risks in humans at the level ordinarily present as impurities.

Tackling impurities through regulation

There are several regulatory guidelines focused on controlling impurities in  pharmaceutical products. Since the guidelines for the ordinary impurities do not specify any acceptable levels for the genotoxic impurities, regulatory authorities have developed ones specifically addressing the genotoxic impurities: “A rationale for determining, testing, and controlling specific impurities in pharmaceuticals that possess potential for genotoxicity”, Muller et al., 2006; “Guideline on the limits of genotoxic impurities”, EMA, 2007; “Questions and answers on the Guideline on the limits of genotoxic impurities’”, EMA, 2010; “Guidance for Industry: Genotoxic and Carcinogenic Impurities in Drug Substances and Products: Recommended Approaches”, FDA, 2008,,; “ Assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic”ICH M7, 2014, and recently published addendum to ICH M7, 2015: “Application of the principles of the ICH M7 guideline to calculation of compound-specific acceptable intakes” (draft).

The purpose of these guidelines is to provide a practical framework that can be applied to the identification, categorization, qualification, and control of genotoxic impurities to limit potential carcinogenic risks.

Toxicological assessments

All actual and potential impurities with known structures that are likely to arise during  synthesis and storage of a drug substance and a drug product should be assessed for their genotoxicity  by literature searches for carcinogenicity and mutagenicity data. If no data are available, an assessment of Structure-Activity Relationship (SAR) should be performed by a computational toxicology assessment (QSAR -quantitative SAR software using one rule-based system (e.g. DEREK) and one statistical-based system (e.g. Sarah) to identify presence of alterting structures in molecules predicting mutagenicity in bacteria. The impurities are then categorized to Classes 1 – 5 (Müller’s approach). Based on the literature data, known mutagenic carcinogens are classified as Class 1, known mutagens with unknown carcinogenic potential as Class 2 and compounds with sufficient effidence of no mutagenic and carcinogenic effects as Class 5. Compounds with the structural alerts, which are not related to the structure of the drug substance, are classified as Class 3 and those with the structural alerts related to the structure of the drug substance which has been proved to be non-mutagenic, as Class 4. Compounds with no structural alerts are Class 5. The outcomes of the computer programs should be always reviewed by experts to provide additional supportive evidence of the results. Since the bacterial mutagenicity assay is able to detect mutagenic carcinogens in rodents and humans, the positive structural alerts may be overruled by negative results of this assay. Optionally, if there are positive results from the in vitro test in bacteria, their relevance can be further investigated using appropriate in vivo tests.

Risk characterization

Appropriate acceptable intakes must be set for the impurities of Class 1 – 3 while the impurities of Class 4 and 5 are treated as non-mutagenic impurities. In case of Class 1 compound-specific limits can be derived either from their rodent carcinogenic potency, if there are positive carcinogenicity data. or their no-observed effect levels if there is evidence of a practical threshold. The compound-specific acceptable intakes can be also based on recommended values published by internationally recognized bodies (e.g. WHO, US EPA).

To control the genotoxic impurities withoutsufficient toxicological data, i.e. Class 2 and 3 compounds,, a concept of Threshold of Toxicological Concern (TTC) has been proposed. The TTC concept defines an acceptable intake of any unstudied chemical that poses a negligible risk of carcinogenicity or other toxic effects. This concept takes into account the fact that duration of exposure is a key factor impacting on the probability of a carcinogenic response. A daily intake of a genotoxic impurity at a level of 1.5 μg/day over life-time is considered to be associated with a negligible carcinogenic risk (<10-5). Accordingly, the acceptable cumulative lifetime dose is 38.3 mg (1.5 μg/day x 25,550 days). Then, if there are less-than-lifetime exposures this cumulative lifetime dose is distributed over total number of days during the exposure. The recommended limits for daily intakes of an individual genotoxic impurities are: 1.5,  10, 20 and 120 μg/day for more than 10 years to lifetime, 1-10 years, 1-12 months and  less than 1 month, respectively. . However, higher intakes may be justified in some cases, e.g. human exposure is much greater from other sources, indications for severe disease, reduced life expectancy, late onset but chronic disease, or with limited therapeutic alternatives etc.

Options to control genotoxic impurities in APIs

The strategy for monitoring genotoxic impurities is based on product and manufacturing process understanding, and utilizes risk management principles, aimed at ensuring process performance and product quality. The impurity may be then tested in the drug substance or raw or intermediate material at or below the acceptable limit, at an intermediate stage with a higher limit if its fate and purge lead to the levels in the drug substance at or below 30 % of the acceptable limit. If the impurity is effectively purged due to its physico-chemical properties and used manufacturing process, no testing of the impurity is required.


Extensive efforts are being made to control impurities, particularly the DNA-reative ones identified based on the toxicity assessments, to assure the safety of the pharmaceutical products. It means extensive investment in resources which may include the redesigning of the synthetic process to avoid introducing of unsafe impurities and/ormodification of relevant process parameters to remove or reduce such impurities to acceptable carcinogenic risk level,  and to take additional considerable measures for controling the impurities at very low concentrations.

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