A Brief Review on Genotoxic impurities in Pharmaceuticals


Dr. Ashok B. Patel1, Ashish H. Asnani1*, Dr. Amitkumar J. Vyas1, Dr. Nilesh K. Patel1,

Dr. Ajay I. Patel1, Dr. Arvind N. Lumbhani2

1Department of Pharmaceutical Quality Assurance, B.K. Mody Government Pharmacy College

Polytechnic Campus, Near Aji dem, Bhavnagar Road, Rajkot - 360003 Gujarat, India.

2Gyanmanjari Pharmacy College, Survey No. 30, Sidsar Road Near Iscon Eleven,

Bhavnagar – 364001 Gujarat, India.

*Corresponding Author E-mail: asnaniashish7@gmail.com



Genotoxic impurities (GTIs) in pharmaceuticals are increasing concern to pharmaceutical industries due to their potential for human carcinogenicity. The literature currently lacks the Guidance for the analytical determination of diverse classes of GTIs. This review article provides brief information regarding Genotoxic impurity (GTI), its sources, their classification, and existing regulatory approaches to control Genotoxic impurities in Pharmaceuticals, also information regarding different types of GTI and examples of each class. It is very difficult for researchers to detect GTI at Trace level so the detection method is also given in the chart, almost all the GTI are Mutagenic but as shown in Nitrosamines it is Mutagenic and its carcinogenicity is also proved but there are no such strong evidence and literature which shows genotoxicity. So, it is included as Mutagenic & carcinogenic. Different Control strategies to control Genotoxicity are also discussed.


KEYWORDS: Genotoxic Impurities (GTI), Toxicity, Classification, Acceptance limits, Mutagenic.




Pharmaceutical impurities are the undesirable chemicals that remain within the active pharmaceutical ingredients (APIs) which are developed during formulation or due to the aging of both API as well as formulated APIs to medicines. The presence of those undesirable chemicals even in the smallest amounts may produce an effect on the safety and efficacy of the pharmaceutical products.



International Council for Harmonisation (ICH) in its guideline ICH S2 (R1) defines genotoxicity as “a broad term that refers to any undesirable change within the genetic material, consideration for the mechanism by which the change is induced.” While genotoxic impurities are defined as “Impurity that has been indicated to be genotoxic in a relevant genotoxicity test model.  E, g., bacterial gene mutation (Ames) test.’’



Impurities Classification for Mutagenic and Carcinogenic Potential and Resulting Control Actions, ICH M7 Draft Guideline is given in table-1.


Table-1: Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk, ICH M7, ICH Harmonised Tripartite Guideline, International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, 2013



Proposed Action for Control


Known mutagenic carcinogens

Control at or below compound-specific acceptable limit


Known mutagens with unknown carcinogenic potential (bacterial mutagenicity positive, no rodent carcinogenicity data)

Control at or below acceptable limits (generic or adjusted TTC)


Alerting structure, unrelated to the structure of the drug substance; no mutagenicity data

Control at or below acceptable limits (generic or adjusted TTC) or do bacterial mutagenicity assay: If nonmutagenic = class 5

If mutagenic = class 2


Alerting structure, same alert in drug the substance that has been tested and is nonmutagenic

Treat as a nonmutagenic impurity


No structural alerts, or alerting structure with sufficient data to demonstrate lack of mutagenicity

Treat as a nonmutagenic impurity



3.1 PhRMA Approach6:

The Pharmaceutical Research and Manufacturing Association (PhRMA) formed a task force in 2004 to discuss testing, classification, qualification, and toxicological risk assessment of PGIs in pharmaceuticals. It instructs that some structurally alerting functional groups are known to be involved in reactions with DNA. These DNA-affecting functional groups were categorized into three groups:

Group 1:

Aromatic groups, for example, N-hydroxyaryls, N-acylated aminoaryls, aza-aryl N-oxides, aminoaryls, and alkylated aminoaryls, purines or pyrimidines, intercalators, etc.,

Group 2:

Alkyl and aryl groups, for example, aldehydes, N-methylols, N-nitrosamines, nitro compounds, carbamates, epoxides, aziridines, propiolactones, propiosultones, beta haloethyl, hydrazines, and azo compounds.

Group 3:

Heteroaromatic groups includes Michael-reactive acceptors, alkyl esters of phosphonates or sulfonates, haloalkenes, primary halides (alkyl and aryl-CH2). PhRMA categorized the impurities into five classes (as mentioned above in table 1)


3.2 USFDA draft guidance6:

In December 2008, the USFDA published draft guidance entitled “Genotoxic and Carcinogenic Impurities in Drug Substances and Products-Recommended Approaches”. Lately, it has been replaced by the ICH M7 guideline on “Assessment and Control of DNA Reactive Impurities (PGIs) in Pharmaceuticals to Limit Potential Carcinogenic Risk.” The USFDA guidelines have provided specific recommendations regarding the safety qualification of impurities with known/suspected genotoxicity. These guidelines describe a variety of ways to characterize and reduce the potential cancer risk associated with the patient's exposure to genotoxic and carcinogenic impurities. The recommended approaches include (i) Prevention of genotoxic and carcinogenic impurities formation, (ii) Reduction of genotoxic and carcinogenic impurities level (maximum daily allowable target of 1.5µg/day), (iii) Additional characterization of genotoxic and carcinogenic risk, and (iv) Considerations for flexibility in approaches to better support appropriate impurity specifications.


3.3 ICH guidelines7-9:

The ICH published guidelines on impurities of drug substances and pharmaceutical products in the late 1990s. In the guidelines, genotoxicity tests have been defined as in vitro and in vivo tests for detecting compounds that induce genetic damage directly or indirectly (International Conference on Harmonization, 1997). The ICH quality guidelines Q3A(R) and Q3B(R) respectively address the topics of control of impurities in drug substances and degradants in pharmaceutical products, while the Q3C guideline deals with the residual solvents.


Table 2: Threshold for APIs:



Maximum daily dose

≤2 g/day

>2 g/day

Reporting threshold



Identification threshold

0.10% or 1.0 mg per day intake (whichever is lower)


Qualification threshold

0.15% or 1.0 mg per day intake (whichever is lower)



3.4 EMEA guideline10-11:

The European Medicines Agency (EMEA) guideline describes a framework and practical approaches on how to deal with genotoxic impurities in new active substances. As per the guideline "The toxicological assessment of genotoxic impurities and the determination of acceptable limits for such impurities in active substances is a difficult issue and not addressed in sufficient detail in the existing ICH Q3X guidance". Also, the EMEA guideline projected a toxicological concern (TTC) threshold value of 1.5 μg/day intakes of a genotoxic impurity which is considered to be associated with an acceptable risk (excess cancer risk of <1 in 100,000 over a lifetime) in most pharmaceuticals. Based on the TTC value, a permitted level of an active substance can be calculated concerning the expected daily dose. Higher limits might be justified under certain conditions such as short-term exposure periods (European Medicines Agency/Committee for Medicinal Products (CHMP) for Human Use, 2006). In the context of this guideline, the classification of a compound (impurity) as genotoxic in general indicates that there are positive findings in established in vitro or in vivo genotoxicity tests with the focus on DNA reactive substances that have a potential for direct DNA damage. In the absence of such information, in vitro genotoxins are usually considered as probable in vivo mutagens and carcinogens (EMEA/CHMP, 2006)


Based on the importance of the mechanism of action and the dose-response relationship in the assessment of genotoxic compounds, the EMEA guideline presents two classes of genotoxic compounds:

1.     Genotoxic compounds with sufficient (experimental) evidence for a threshold-related mechanism,

2.     Genotoxic compounds without sufficient (experimental) evidence for a threshold-related mechanism.



Mainly Genotoxic impurity comes from the starting material products and intermediate reaction. The main sources are shown in fig.1.


Figure 1: Sources of genotoxic impurity



A control strategy is planned for current product and process understanding that gives assurance that the process performance and product quality free of GTIs (ICH Q10). A control strategy includes the following options:

·       Controls on material attributes (including raw materials, starting materials,

·       Intermediates, reagents, solvents, and primary packaging materials)

·       Facility and equipment operating conditions

·       Controls implicit in the design of the manufacturing process

·       In-process controls (including in-process tests and process parameters)

·       Controls on drug substance and drug product (e.g., release testing).


The FDA draft guidance was generally quite similar to the EMEA guideline.


Some notable exceptions are:

·       The FDA includes carcinogenic impurities (many carcinogens are non-genotoxic)

·       The FDA includes additional safety margins for pediatric populations.


Control strategy 1 – avoidance:

According to the EMEA decision tree in the 2006 guideline, during production avoidance of side reaction and formation of reactive molecules which may act as GTIs can be a control strategy.


eg: Various reaction involved acids with alcohols and can produce various alkylating agents such as alkyl halides, esters of aryl sulfonic acids (besylate and tosylates and esters of sulfuric acid), and esters of alkyl sulfonic acids (mesylates such as ethyl mesylate (EMS) and m EMS) all are reported to be potential GTIs. Such cases can be avoided by substituting such acids with alternative acids so that the production of GTIs is avoided.

Control strategy 2 – adjust API process:

It is more difficult to adjust the optimal chemical process of API which leads to the formation of GTIs. But by applying some strategies, we can control over GTIs. Examples are:

·       Placing GTIs early in the process so that the produced GTI will be available longer duration for purging and will remain away from API forming step so that the GTI can be effectively controlled.

·       Placing solid isolations strategically in the process such as crystallization for the purification of intermediates and APIs, will help in a reduction or remove crucial GTIs.


Control strategy 3 – demonstrate DTI threshold mechanism above TTC level:

High levels of Ethyl methane sulphate (EMS), a potential GTI, lead to a recall of Roche’s Viracept (nelfinavir mesylate) in 2007. It was due to improper cleaning of vessels with ethanol for a longer duration of time and further deposition of EMS.


This cause was eventually traced to a GMP failure in the manufacture of the API. While Roche’s investigation on in vivo rodent toxicology studies with EMS and threshold of 2mg EMS/kg for DNA damage was determined. It was four orders of magnitude higher than the 1.5µg/day TTC as per EMEA guidelines. Based on this finding, the EMEA accepted a higher TTC for EMS. It has been speculated that this result may ultimately provide a new approach to guiding risk management for genotoxic impurities in pharmaceuticals.



6.1 Alkyl halides:

Methyl, ethyl, and propyl halides are used widely as industrial alkylating agents. Although the mechanism of their toxicity is still not fully understood, they have been shown to directly alkylate critical biologically active macromolecules, such as proteins and DNA. The source of an alkyl halide in APIs streams can be derived not only from the direct use of alkyl halides but also from side reactions between alcoholic solvents and hydrogen halides or dequaternization of ammonium salts.13


The synthesis of fexofenadine, an antihistaminic agent, involves the base-catalyzed C-methylation of 4-bromophenylacetonitrile with genotoxic methyl iodide, yielding the dimethyl derivative, as illustrated in a similar reaction, an intermediate in the synthesis of the bis-acetonitrile aromatase inhibitor anastrozole is submitted to exhaustive alkylation using sodium hydride and methyl iodide.


Figure 2: C-Alkylation Step in the Synthesis of (a) Fexofenadine or (b) Anastrozole Can Result in Traces of Genotoxic Methyl Iodide


6.2. Dialkyl Sulfates:

The most common dialkyl sulfates used in the pharmaceutical industry are the methyl and ethyl derivatives, the latter having been used as a chemical weapon. Being a strong methylating agent, dimethyl sulfate (DMS) is used to introduce a methyl group to atoms featuring unshared electron pairs, such as oxygen, nitrogen, carbon, sulfur, phosphorus, and some metals. Compared with the alkyl halide-type methylating agents, DMS is more favorable due to the higher reaction rate and lower possibility of by-product formation.


In the quaternization of an aromatic amine during the last synthetic step of neostigmine, a parasympathomimetic drug that acts as a reversible acetylcholinesterase inhibitor. Dimethyl sulfate is used in the last step of the API synthesis to form the quaternary ammonium salt, neostigmine14


Figure 3: Quaternary Ammonium Salt Formation using Dimethyl Sulfate


6.3. Epoxides:

Epoxides are the simplest cyclic ethers, featuring three ring atoms. Due to the large ring strain associated with the three-membered ring, epoxides are highly reactive molecules and thus are often used as reagents in API manufacturing. They easily participate in epoxide-ring-opening reactions with alcohols, amines, halides, organometallics, cyanides, sulfides, aromatic compounds, and active methylene groups. On the other hand, the high reactivity of these compounds makes them genotoxic, as their two electrophilic carbon atoms can react with the DNA nucleophilic centers, giving alkylated products. Substituted epoxides, such as 2,3- epoxypropanol (glycidol), 1-chloro-2,3-epoxypropane (epichlorohydrin), or 1,2-epoxy-3-butene, are often used as building blocks during the synthesis of APIs. They are often subjected to epoxide-ring-opening reactions, and since they are bifunctional, they usually act as linking agents or can form heterocycles.15


An intermediate of the antiretroviral drug darunavir is prepared using phenylmagnesium bromide and commercially available 1,2-epoxy-3-butene in the presence of catalytic CuCN to furnish the corresponding allylic alcohol.


Figure 4: Use of 1,2-Epoxy-3-butene during the Synthesis of Darunavir


6.4. Hydrazines:

The toxicity of hydrazine and its derivatives is ascribed to the generation of carbocations, carbon-centered radicals, and oxygen-centered radicals, which are considered to be highly reactive species. For these reactive intermediates, DNA alkylation and other DNA lesions have been reported.16


The preparation of sildenafil, which is generally known as Viagra, a drug used for the treatment of erectile disfunction, involves the hydrazine-assisted formation of a substituted pyrazole ring.17


A similar reaction takes place during the synthesis of the topoisomerase inhibitor sedoxantrone. One of the steps of sedoxantrone synthesis is the condensation of a phenol intermediate with substituted hydrazine, which leads to pyrazole formation. Though the order of the reaction steps has not been established, the formation of the hydrazone, then displacement of the adjacent chlorine by the second nitrogen, and final closure of the pyrazole ring seems plausible.


Figure 5: Pyrazole Formation with Hydrazine during the Preparation of (a) Sildenafil and (b) Sedoxantrone


6.5. TEMPO:

2,2,6,6-Tetramethylpiperidin-1-oxyl free radical (trade name TEMPO) is a commonly employed process reagent and potential process impurity. This compound was evaluated for genotoxic potential, and based on the available, and somewhat conflicting, published data, it is considered to be genotoxic. TEMPO is widely used throughout chemical- and biochemistry-related industries as stable nitroxyl radical. TEMPO is mainly used for oxidations of alcohols to yield aldehydes and ketones or carboxylic acids.18


Oseltamivir is a neuraminidase inhibitor and is the most commonly prescribed drug for treatment to combat influenza. The large number of synthetic approaches reported in the literature implicates the importance of this drug. In the example below, TEMPO is used with trichloroisocyanuric acid (TCCA) in the oxidation of a secondary alcohol to give the corresponding ketone intermediate.19


Figure 6: TEMPO-Assisted Oxidation of a Secondary Alcohol Intermediate to a Ketone during Oseltamivir Synthesis


6.6. Aromatic Amine:

Although aromatic amines are generally not inherently genotoxic, during metabolic activation, electrophilic species are generated. The main transformation pathway of aromatic amine metabolism is oxidation, producing an N-hydroxy compound that is conjugated as an acetate, sulfate, or glucuronide. Further deconjugation results in a nitrenium ion (ArN+H), which is considered to be the active genotoxin that binds to DNA.20


Chlorhexidine was discovered more than 60 years ago and since then it has been used in more than 60 pharmaceuticals and medical devices. It is widely used as a disinfectant and topical antiseptic and has found applications in catheters and preoperative skin preparations. As shown in the final step of its synthesis involves the use of potentially genotoxic 4 -chloroaniline.


Figure 7: Use of Potentially Genotoxic 4-Chloroaniline in the Final    Synthetic step of chlorhexidine


6.7. Boronic Acids:

Boronic acids have been recently tested and identified as a novel family of bacterial mutagens. However, there is no direct evidence of direct covalent binding between them and DNA. Twelve out of the 13 boronic acid derivatives recently tested by O’Donovan et al. were shown to be mutagenic.21


Angiotensin II receptor antagonist losartan is used for the treatment of hypertension. The synthetic step involving a Suzuki coupling in the synthesis of losartan developed by Merck research chemists is outlined in the following figure. First, the trityl-protected phenyl tetrazole was ortho lithiated using butyllithium and then quenched with tri-isopropyl borate, giving the boronic acid derivative after treatment with aqueous ammonium chloride. The resulting boronic acid participated in a Suzuki cross-coupling reaction with the other key intermediate, imidazole alcohol.


Figure 8: Suzuki Coupling during the Merck Process for the Synthesis of Losartan


6.8 Sulfonates Esters and Their Precursors:

Sulfonate esters are alkylating agents, a class of potentially genotoxic compounds. They are called alkylating agents due to their ability per se, or after metabolic activation, of adding alkyl residues to the reactive nucleophile sites of the DNA bases.22


Figure 9: Common Sulfonate Ester Impurities and Their Precursors


Due to their synthetic versatility, sulfonate derivatives are common and useful reagents in the pharmaceutical industry, especially in reactions where carbonium ion initiation is needed. Examples of such compounds are mesylates (methane sulfonate), triflates (trifluoromethanesulfonate), tosylates (p-toluene sulfonate), nosylate (4-nitrobenzenesulfonate), and besylates (benzenesulfonate). Sulfonate ester impurities may be present in APIs or their intermediates (i) due to their production inside reactions between sulfonic acids or halides and alcohols or (ii) as reactants carried over from incomplete reactions.


Sulfonic acids are salt-forming agents used in the last step of the API synthesis. Basic APIs are usually preferentially presented in the salt form due to their higher aqueous solubility and subsequently higher bioavailability. The conversion of an API to salt also can help to enhance stability and water solubility and helps isolation as final product Elder et al. overviewed the utility, safety, and regulation of APIs formulated as sulfonic acid salts. For example, methane sulfonic acid is used in the production of Viracept.23


Figure 10: Final Manufacturing Step of Viracept Drug Substance Nelfinavir Mesylate


6.9 Nitrosamines*:

Nitrosamines, or N-nitrosamines, is any molecule that contains the (N–NO) nitroso functional group. N-Nitrosamines are polar hydrophilic, uncharged molecule which has high vapor pressure and very high solubility in water. N -nitroso compounds are a large group of potent carcinogens which includes N-nitrosamine. These N- Nitrosamines are derived from an alkyl, alkaryl, aryl, or cyclic amines. It is a group they share with N-nitrosamides which are derived from N-alkylureas, N- alkyl carbamates, and simple N-alkyl amides. These compounds are related to nitrosamine impurities and that nitrosamine impurities are probable human mutagenicity, teratogenicity, and carcinogenicity.24


ICH M7 (R1) classifies Nitrosamine impurities as Class 1 Genotoxic impurities, which are known to be mutagenic and carcinogenic, based on both rodent carcinogenicity and mutagenicity data. These Nitrosamine impurities produce an effect on the genetic material using mutations through chromosomal breaks, rearrangements, covalent binding, or insertion into the DNA during replication. These changes within the genetic materials produced by the exposure to very low levels of Nitrosamine impurities can result in cancer. Thus, it’s important to identify Nitrosamine impurities in drugs at very low levels to make sure safety to the general public.25

A total of 10 nitrosamine impurities is reported for mutagenic potential

1.     N-nitrosodimethylamine

2.     N-nitrosodiethylamine

3.     N-nitroso-methyl ethylamine

4.     N-nitrosodi-n-propylamine

5.     N-nitrosodiisopropylamine

6.     N-nitroso di-n-butylamine

7.     N-nitrosodiphenylamine

8.     N-nitrosopyrrolidine

9.     N-nitrosopiperidine

10. N-nitrosomorpholine



Figure 11: Structure of Nitrosamine Impurities


7. Identification, Control, and Determination of GTIS In Drug Substances. 26-29


Figure 12: Flow diagram for Identification, Control, and Determination of GTIS In Drug Substances.



The present review article describes what is genotoxic impurity (GTI), its sources, its classification, and existing regulatory approaches to control Genotoxic impurities in Pharmaceuticals, also this review article gives brief information regarding different types of GTI and covers examples of each class. It is very difficult for researchers to detect GTI at Trace level so the detection method is also mentioned in the chart, almost all the GTI are Mutagenic but as shown in Nitrosamine it is Mutagenic and its carcinogenicity is also proved but there are no such strong evidence and literature which shows genotoxicity. So, it is included as Mutagenic & carcinogenic. Also, different control strategies were discussed to reduce the GTI in the pharmaceuticals.



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Received on 22.03.2021            Modified on 10.05.2021

Accepted on 05.06.2021   ©Asian Pharma Press All Right Reserved

Asian J. Pharm. Res. 2021; 11(3):187-193.

DOI: 10.52711/2231-5691.2021.00034