2. Carcinogenic Impurity:

2.1. NDMA (N-nitroso dimethylamine):

NDMA (N-nitroso dimethylamine) is a member of the “N-nitrosamine” class. According to IARC, it is classified in the class of GROUP 2A probably carcinogenic to humans.⁸ NDMA is a hepatotoxic and genotoxic substance.⁹

NDMA is defined as a chemical substance that belongs to the class of potent carcinogens and, as per the US Department of Health and Human Services; exposure to a high amount of NDMA may lead to liver damage. This is confirmed by animal studies that indicate that NDMA can create tumors in the kidney, liver, and respiratory tract, making it a likely human carcinogen.¹⁰

The generic chemical structure of NDMA is R₂N–N=O, a deprotonated amine attaches to a nitroso group. N-nitrosamines are generally created when a secondary or tertiary amine reacts with a nitrosating agent.¹¹

2.1.1. Structure of impurity and drug in which formed:

2.1.2 Source:

NDMA contamination may occur in some medications. There are two sources of NDMA contamination in medicine.

1. The first source of contamination is the use of NDMA contaminated material during the manufacturing process of medication.

2. The second source of NDMA contamination is NDMA produced from an intermediate or API (active pharmaceutical ingredient) itself. Ex, Ranitidine contains both nitrite and diethylamine group which create NDMA under stomach relevant pH.⁹

In the past two years, NDMA was found in some drugs like ranitidine, some sartan class drug-like valsartan, and metformin. Due to the presence of this impurity this all drug was removed from the market.¹⁰

2.1.3 Formation pathway of NDMA in ranitidine:

In September 2019, the US Food and Drug Administration (FDA) reported routine testing had detected N-nitrosodimethylamine (NDMA) impurity in ranitidine.¹² The researchers documented that the formation of NDMA by nitrosation of ranitidine in stomach-relevant pH conditions in vitro and produced a mechanism of NDMA formation from ranitidine.¹³

Figure 2: Mechanism of NDMA formation from ranitidine.

Decomposition of ranitidine raise with raising NaNO₂-to-ranitidine molar ratios. Previous research has documented that nitrosation of tertiary amines, such as the dimethylamino-carrying group in ranitidine, forms an unstable tertiary N-nitrosamine. This unstable intermediate can undergo dealkylation to form DMA, which is also undergoing nitrosation to form NDMA, a stable secondary N-nitrosamine.¹⁴

As the figure shows, NDMA is creating from the nitro and dimethylamino groups present in the drug structure, and Cl^– and H⁺ play main roles in the conversion of these groups into NDMA. The formation of nitrosyl chloride, which is a potent nitrosating agent, is also a critical step.¹⁵

Figure 3: Decomposition of ranitidine producing NDMA¹⁴

2.1.4 Formation of NDMA in sartans:

In July 2018 FDA and EMA announced that carcinogenic impurity NDMA was found in some generic drugs especially in “sartan” class drugs.¹⁶

Figure 4: Sartan drugs with tetrazole ring

Sartan contains a tetrazole ring in their structure, during the synthesis of a tetrazole ring NDMA is created in presence of dimethylformamide (DMF) reagent. DMF is used as a solvent; it can decompose under certain conditions and create small amounts of dimethylamine (DMA). In acidic conditions, this amine then reacts with another chemical used in the manufacturing process called sodium nitrite (NaNO₂), and in the acidic condition, it formed NDMA.¹⁷

Figure 5: Decomposition of DMF to form NDMA¹⁷

2.1.5 Formation of NDMA in valsartan:

Valsartan belongs to the class of angiotensin II receptor antagonists. It is used in the treatment of hypertension, congestive heart failure (CHF), and myocardial infarction.^16,18

The presence of N-nitrosodimethylamine (NDMA) in valsartan tablets may lead to a voluntary product recall of valsartan finished formulations in some industries. It was found that a change in the manufacturing process of valsartan API may lead to the formation of this impurity.¹⁹

According to figure no. 6 synthesis of valsartan starts from (S)-valin methyl ester and 4-(bromomethyl)-[1,1_-biphenyl]-2-carbonitrile or 2-cyano-4-formylbiphenyl. As final step of the synthesis is the formation of the tetrazole moiety by reaction with azidotributyltin (IV).²⁰

ZHP (Zhejiang Huahai Pharmaceuticals) made a change in their manufacturing process to reduce waste and improve yield. ZHP changed the synthetic process of tetrazole ring formation in the valsartan. Tributylin azide was replaced with a more toxic compound sodium azide, and dimethyl formamide was used as solvent. Sodium nitrite was then used to quench excess sodium azide in synthesis. In the acidic environment, sodium nitrite creates nitrous acid, which could react with dimethylamine present in dimethylformamide (the solvent used in tetrazole forming reaction) to produce NDMA.²⁰

Figure 6: Synthesis pathway of valsartan according to European pharmacopeia.²⁰

Figure 7: Tetrazole ring formation with sodium azide.²¹

2.1.6 Metabolism of NDMA:

NDMA requires metabolic activation to produce their carcinogenic and mutagenic effect.²² NDMA is primarily metabolized by CYP2E1 in the liver of rodents and then leading to the formation of a methyl diazonium ion. After that this methyl diazonium ion reacts with DNA and predominantly creates N⁷ and O⁶ -methyl guanine (N⁷MG and O⁶ MG), the latter being highly mutagenic.¹⁸

Figure 8: Metabolic activation of NDMA to form DNA reactive methyl diazonium ion.

NDMA is metabolized by CYP2E1 and formed methyl diazonium ion by α-hydroxylation pathway.^23,24 Toxicological effect of NDMA depends on metabolic activation by CYP2E1 and DNA-repair capacities for the specific DNA-repair adducts form.¹⁸

The primary DNA adducts formed following exposure to NDMA is N⁷ -methylguanine and O⁶ -methylguanine is a secondary adduct. Other DNA adducts created in smaller amounts include N³ -methyladenine, and O⁴ -methylthaymine.

The formation and persistence of the secondary adduct, O⁶ -methylguanine, being associated with both the carcinogenicity and mutagenicity of NDMA.²⁴

2.2.1 NDEA (N-nitrosodiethylamine):

NDEA (N-nitrosodiethylamine) belongs to the “N-nitrosamine” class.¹⁰ As per IARC, this impurity is classified under class group 2A, probably carcinogenic to humans.⁸

2.2.2 Structure:

Figure 9: Structure of NDEA

FDA and EMA in July 2018, announced that carcinogenic impurity NDMA is found in some generic sartan class drugs include valsartan. In November 2018 irbesartan is recalled from the market due to NDEA impurity found.¹⁶

2.2.3 Formation of NDEA in valsartan:

Valsartan belongs to sartan class this class compound contains tetrazole ring in their structure. During tetrazole ring formation NDEA is formed as a byproduct. NDEA may be generated from DEA (diethylamine) as same as NDMA formed from DMA.¹⁸

Figure 10: Formation of NDEA from diethylamine.

DEA could be formed from the degradation of triethylamine (TEA) or present as an impurity in the raw material of TEA.¹⁸

Figure 11: Formation of NDEA from TEA by nitrosation.

Direct nitrosation of TEA may cause by nitrosonium ion, resulting in the formation of an aldehyde and a secondary amine,which further react with nitrous acid to form a nitrosamine.¹⁸

2.2.4 Metabolism of NDEA:

NDEA requires metabolic activation to give their carcinogenic effect. Metabolism of NDEA is similar to NDMA with the primary activation step by α-hydroxylation to α-hydroxyl-NDEA and finally creating an ethyldiazonium ion reacting with DNA and formation of O⁴ -ethyldeoxythymidine (O⁴ ET) and O⁶ -ethylguanin (O⁶ EG) which are primary mutagenic adducts.¹⁸ This DNA adducts formation depends on the individual negative charge present in each DNA base.²⁵

NDEA is metabolized in humans primarily by enzyme CYP2E1 and CYP2A6. NDEA causes a raise in mutation in the different organ of mouse and cause cancer in different species and different organ of a laboratory animal in which the liver is being the primary target organ.¹⁸

The toxicological effect of NDEA depends on metabolic activation by CYP2E1 and/or CYP2A6 and DNA-repair capacities for the specific DNA-adducts formed. NDEA is biotransformed to create an ethyl diazonium ion by cytochrome p450 enzymes (mainly CYP2E1). Ethyl diazonium ion can further react with nucleophilic sites of DNA and form adducts which can induce cancer.¹⁹

Figure 12: Biotransformation of NDEA and mechanism of DNA-adduct formation.

2.3.1 NMBA (N-nitroso-N-methyl-4-aminobutyric acid):

NMBA belongs to the class of N-nitrosamine. The impurity is a known animal and potential human carcinogen. N-Nitroso-N-methyl-4-aminobutyric acid (NMBA) is a tobacco-specific nitrosamine carcinogen. It is oxidized to the reactive metabolite methyl-2-oxopropylnitrosamine (MOPN) in isolated rat liver mitochondria. NMBA produces bladder transitional cell carcinomas in rats when administered in the drinking water.²⁶

2.3.2 Structure of NMBA:

Figure 13: Structure of NMBA

2.3.3 Formation of NMBA:

NMBA could be formed during the synthesis of losartan while using sodium nitrite and N-methyl pyrrolidone.¹⁸

Figure 14: Formation of NMBA

Formation of NMBA is attributed to the generation of 4-(Methylamino) butyric acid due to the hydrolysis of NMP and excess of NaNO₂ and HCl, used for quenching the excess Sodium azide / HN₃.²⁷

Limits of nitrosamine impurity:

In European Union, EMA gave temporary interim limits for nitrosamine impurity in sartan.^28,29

Table 4: EMA published limits for nitrosamine in sartans.

Drug	MD (mg/day)	Acceptable intake
Drug	MD (mg/day)	NDMA (ng/day)	NDMA (ppm)	NDEA (ng/day)	NDEA (ppm)	NMBA (ng/day)	NMBA (ppm)
Valsartan	320	96.0	0.300	26.5	0.082	96.0	0.300
Losartan	150	96.0	0.640	26.5	0.177	96.0	0.640
Olmesartan	40	96.0	2.400	26.5	0.663	96.0	2.400
Irbesartan	300	96.0	0.320	26.5	0.088	96.0	0.320
Candesartan	32	96.0	3.000	26.5	0.820	96.0	3.000

In the United States, interim limits for nitrosamine impurity in sartan are developed and published by the FDA.^28,30

Table 5: FDA published an interim limit for nitrosamine in sartans.

Drug	MD (mg/day)	Acceptable intake
Drug	MD (mg/day)	NDMA (ng/day)	NDMA (ppm)	NDEA (ng/day)	NDEA (ppm)	NMBA (ng/day)	NMBA (ppm)
Valsartan	320	96	0.3	26.5	0.083	96	0.3
Losartan	100	96	0.96	26.5	0.27	96	0.96
Irbesartan	300	96	0.32	26.5	0.088	96	0.32
Azilsartan	80	96	1.2	26.5	0.33	96	1.2
Olmesartan	40	96	2.4	26.5	0.66	96	2.4
Eprosartan	800	96	0.12	26.5	0.033	96	0.12
Candesartan	32	96	3.0	26.5	0.83	96	3.0
telmisartan	80	96	1.2	26.5	0.33	96	1.2

MD*= maximum daily dose

2.4.1 MNP (1-methyl-4-nitrosopiperazine):

MNP (1-methyl-4-nitrosopiperazine) belongs to the nitrosa mine class of compounds, some of which are classified as probable or possible human carcinogens (substances that could cause cancer), based on laboratory tests such as rodent carcinogenicity studies. Although there are no data available to directly evaluate the carcinogenic potential of MNP, information available on closely related nitrosamine compounds was used to calculate lifetime exposure limits for MNP.³¹

2.4.2 Structure of MNP and drug in which formed:

Figure 15: Structure of MNP and drug in which formed.

2.4.3 Impurity in rifampin:

FDA recently became aware of nitrosamine impurities found in certain samples of rifampin which was MNP (1-methyl-4-nitrosopiperazine).³² To avoid a potential shortage of this drug, the FDA is allowing manufacturers to temporarily distribute products with impurities above the acceptable intake limits while an investigation into the source continues. FDA and manufacturers are investigating the cause of this impurity in rifampin.³³

2.4.4 Limits:

As per the FDA, the acceptable intake limits are 0.16 parts per million (ppm) for MNP in rifampin. The agency will not object to certain manufacturers temporarily distributing rifampin containing MNP below 5 parts per million (ppm).³¹

2.5.1 CPNP (1-cyclopentyl-4-nitrosopiperazine):

CPNP belongs to the nitrosamine class of compounds, some of that are classified as probable or possible human carcinogens (substances that could cause cancer), based on laboratory data such as rodent carcinogenicity studies. Currently, there is no evidence available to check the carcinogenic potential of MNP and CPNP.³¹

2.5.2 Structure of CPNP and drug in which it formed:

Figure 16: Structure of CPNP and drug in which formed

2.5.3 CPNP in rifapentine:

FDA recently became aware of nitrosamine impurities found in certain samples of rifapentine which was CPNP (1-cyclopentyl-4-nitrosopiperazine).³² To avoid a potential shortage of this drug, the FDA is allowing manufacturers to temporarily distribute products with impurities above the acceptable intake limits while an investigation into the source continues. FDA and manufacturers are investigating the source of this impurity in rifapentine.³³

2.5.4 Limits:

The acceptable intake limit is 0.1 ppm for CPNP in rifapentine. The agency also will not object to certain manufacturers temporarily distributing rifapentine containing CPNP below 14 ppm.³¹

2.6.1 DMF (dimethylformamide):

Dimethylformamide is belongs to the class of formamides that is formamide in which the amino hydrogens are replaced by methyl groups. It has a role as a polar aprotic solvent and a hepatotoxic agent. It is a volatile organic compound and belongs to the formamide class. It derives from a formamide.³⁴ DMF is a chemical that was reclassified in 2018 as a Group 2A probable human carcinogen by the World Health Organization (“WHO”) and (“IARC”).³⁵

2.6.2 Structure:

Figure 17: Structure of DMF

FDA has been informed that a solvent called dimethylformamide (DMF) was discovered in valsartan made by several companies, including Novartis. DMF is classified by the World Health Organization as a probable carcinogen.³⁶ If DMF were present in valsartan that also contained NDMA, which could exacerbate the cancer risks, according to Valisure. Its tests detected DMF in valsartan alone and in pills where it was combined with a blood-pressure drug called hydrochlorothiazide.³⁷

2.6.3 Metabolism of DMF:

Figure 18: Biotransformation of DMF

The major metabolic pathway for DMF in mammalian species is oxidation by the cytochrome P-450- dependent mixed-function oxidase system to HMMF. This can generate NMF and formaldehyde. Further cytochrome P450-mediated oxidation of NMF and/or HMMF results in the formation of S-(N-methyl carbamoyl) glutathione (SMG), the conjugate of the presumed reactive (toxic) intermediate, methyl isocyanate, excreted in vivo as N-acetyl-S-(N-methyl carbamoyl) cysteine (AMCC).³⁷ The proposed mechanism of the key step in the metabolic activation of N, N-dimethylformamide through the oxidation of N-methyl formamide is shown in Figure 19. Methyl isocyanate was also reported to be the main product of N-methyl formamide photo-oxidation with hydroxyl radicals under aerobic conditions and the carbonyl centered radical was proposed as a precursor to methyl isocyanate based on quantum chemical calculations.^37,38

Figure 19: Proposed mechanism of generation of the key electrophilic carbamoylating species, methyl isocyanate

2.7 Ethyl methanesulfonate:

Ethyl methanesulfonate is alkyl sulfonic acid salt, it is also known as ethyl mesylate.³⁹ According to IARC EMS is classified as group 2B possibly carcinogenic to humans.⁸ Ethyl methanesulfonate is a methanesulfonate ester resulting from the formal condensation of methanesulfonic acid with ethanol. It has a role as an alkylating agent, an antineoplastic agent, a carcinogenic agent, a genotoxin, a mutagen, and a teratogenic agent.⁴⁰

2.7.1 Structure of EMS and drug in which formed:

Figure 20: Structure of EMS and Viracept

2.7.2 EMS in Viracept:

On 5 June 2007, the marketing authorization holder (MAH) informed the European medicines agency (EMA) that Viracept was being recalled from European Union markets with immediate effect since contamination of the product with a genotoxic substance (ethyl methanesulfonate; ethyl mesylate; EMS) had been detected.⁴¹

2.7.3 Formation:

Figure 21: Formation of alkyl sulfonates from sulfonic acid and short-chain aliphatic alcohol.

The root cause of the contamination was a GMP failure in respect of the holding tank for MSA, which is used in the final step of the synthesis to convert the nelfinavir base to its mesylate salt. Following non-routine maintenance, the holding tank was cleaned according to the standard operating procedure (i.e. with ethanol), but, crucially, no tank drying was performed. On refilling of the tank with neat MSA, a reaction with the residual ethanol ensued, leading over time (several months) to the production of significant concentrations of EMS. This contaminated MSA was used for the October 2006 campaigns for the manufacture of nelfinavir mesylate API and subsequent drug product batches.⁴⁰

2.7.4 Carcinogenic mechanism:

Ethyl Methanesulfonate is a sulfonoxyalkane with carcinogenic and teratogenic properties. Ethyl methanesulfonate ethylates DNA, thereby damaging DNA and leading to genetic mutations, single-stranded breaks in DNA, and chromosomal aberrations. Ethyl methanesulfonate may be used experimentally in biomedical research.³⁹

3. Analytical Method for Detection of Impurities:

Analytical methods are considered to be a backbone of the pharmaceutical industry and research development laboratories to produce quality products needed for human welfare. Depending on the method of working, chemical analysis can be categorized into two terms as qualitative and quantitative. Qualitative deals with the identification, and quantitative deals with the quantification of desired bioactive/impurity components, respectively. Various analytical methods including FT-IR, UV, HPLC, LC-MS/MS, GC-MS/MS with Headspace, NMR, LC-HRMS, LC-NMR, CE-MS, CE-NMR, SFC-MS, and LC-FT-IR are regularly used nowadays for qualitative and quantitative estimation of pharmaceuticals.⁴² Analytical method validation is required to develop new process, new molecules, active ingredients, residues, impurity profiling and component of interest in different matrices.⁴³

3.1 GC-MS/MS:

Gas chromatography along with mass spectrometry (GC-MS) is the most frequently employed method for the determination of Nitrosamines with lower molecular weight. The majority of recent publications employed GC-MS, GC–HS/MS or GC-MS-MS methods due to its high selectivity and low detection limit.¹⁶

GC-MS is a destructive and hard ionization method for a qualitative and quantitative calculation for volatile organic compound or APIs, Although GC with various detectors can be used for nitrosamine detection, however, nitrogen-phosphorous detector (NPD) and nitrogen chemiluminescence detector (NCD) are the most convenient for the same. In general, NDMA studies are performed in GC-MS using d⁶-NDMA as the internal standard.⁴⁴ Gas chromatography coupled with mass spectrometry is particularly useful in the determination of organic volatile impurities. In this method the carrier gases are such as hydrogen, helium, nitrogen and argon used as mobile phase for the chromatographic separation.^45,46

FDA developed the combined OTR GC/MS headspace method was for the simultaneous assessment of four nitrosamine impurities in ARB drug substance and drug product.

3.2 Liquid Chromatography-Tandem Mass Spectrometry (LC/MS, LC/MS/MS):

LC method offers a faster alternative to the traditional GC-MS methods. The use of high-resolution accurate mass spectrometry helps to obtain good selectivity for the detection of both GC detectable and GC-undetectable compounds along with thermally stable and unstable Nitrosamines.^42,47

LC-MS is a hyphenated method, which separates and quantifies the components from a complex mixture with the help of mass spectrometer. Mass spectrometer separates and estimates the charged components. LC-MS is a well-known method to analyze large, polar, ionic, nonvolatile, and unstable organic compounds. LC-MS/MS analysis is being done via soft ionization and impurity analysis using various types of modern ionization sources including electrospray ionization (ESI), matrix-assisted laser desorption ionization (MALDI), APPI, APCI, CI, EI, FAB, SIM5, Z Spray, and TSP. ESI and APCI are widely used for the analysis of nitrosamine impurities present in various samples such as food materials, drinking water, and pharmaceuticals samples.^42,48

3.3 Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS):

In October 2019, the US Food and Drug Administration (FDA) noted that a third-party lab used higher temperatures in its tests to check for nitrosamine impurities. NDMA was formed by the heat application, but higher temperatures were recommended for the use of a gas chromatography-mass spectrometry (GC/MS) technique to test NDMA on valsartan and angiotensin II receptor blockers (ARBs). The FDA has declared that it recommends the use of a mass spectrometry high-performance liquid chromatography (LC-HRMS Chromatography-High Resolution -Liquid Mass Spectrometry) test protocol to test ranitidine samples. Its LC-HRMS test method doesn't use highs temperatures and showed much lower levels of NDMA in ranitidine drugs than those reported by the third-party laboratory.⁴⁹

A LC-HRMS method was developed and validated following ICH Q2 (R1) for the detection and quantitation of six nitrosamine impurities in losartan drug substance and drug product, including N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitroso-ethyl isopropylamine (NEIPA), N-nitrosodiisopropylamine (NDIPA), N-nitrosodibutylamine (NDBA) and N-nitroso-N-methyl-4-aminobutyric acid (NMBA).

3.4 LC-ESI-HRMS:

In May 2020, FDA received a report from an applicant holder that a single lot of rifapentine drug substance contained 1-Cyclopentyl-4- Nitrosopiperazine (CPNP). OTR had been assigned to develop and validate a method and test for MNP in rifampin and CPNP in rifapentine drug product and drug substance to determine the level of these two nitrosopiperazine impurities. MNP and CPNP belong to the nitrosamine class of compounds, some of which are classified as probable or possible human carcinogens.

A combined LC-ESI-HRMS method was developed and validated according to ICH Q2 (R1) for the estimation and quantitation of MNP in rifampin and CPNP in rifapentine drug substance and drug product.⁵⁰

3.5 HPLC:

Liquid chromatography is a very important separation method that has a significant impact in the area of pharmaceuticals and chemistry. The last decade is the witness where HPLC has played an important role to purify components from the biological complex mixtures. Chromatography is based on the partitioning (relative solubilities in both phases) of solutes between two phases. Various HPLC methods have been reported for nitrosamine impurities. HPLC is a modern analytical technique, and the same is being widely used for formulation, and quality control of nitrosamine impurity estimation in routine daily practice.The HPLC is the most simple, economic, reliable and worldwide used technique in the pharmaceutical analysis.^42,51,52,53

3.6 Rapid-fire MS/MS:

Nitrosamine impurities are believed to have been introduced into the finished products through several pathways that include synthesis and manufacturing routes. OTR has developed an advanced analytics robotics-tandem mass spectrometry method (RapidFireMS/MS) to screen and quantitate the presence of NDMA/NDEA/NMBA nitrosamine impurities in losartan potassium API. The method can be adapted to quantitate these nitrosamine impurities in other “sartan” drug API and products.

A novel RapidFire-MS/MS method has been developed to simultaneously quantify NDMA, NDEA, and NMBA in losartan potassium API.⁵⁴

4. CONCLUSION:

In pharmaceutical industry many drugs are contaminated with carcinogenic impurity. Because of their carcinogenic effect these drugs are recalled from market for safety of consumer. In past two-year nitrosamine class impurity are found in many drugs and which are recalled from market. NDMA, NDEA and NMBA is belongs to nitrosamine class which are probable carcinogenic to human. It is formed in sartan class drugs which contains tetrazole ring in their structure. NDMA and NDEA are metabolized by α-hydroxylation pathway and create DNA-adducts. NMBA could be formed during the synthesis of losartan while using sodium nitrite and N-methyl pyrrolidone. MNP and CPNP are nitrosamine impurities formed in rifampin and rifapentine which are antituberculosis drugs. FDA has been informed that a solvent called dimethylformamide (DMF) was discovered in valsartan made by several companies. DMF has classified by WHO as probable human carcinogen. The major metabolic pathway for DMF in mammalian species is oxidation by the cytochrome P-450- dependent mixed-function oxidase system to HMMF. Ethyl methanesulfonate is alkyl sulfonic acid salt which are possible human carcinogen found in viracept drug produce due to GMP failure. Various analytical method are used for analyze and quantify these carcinogenic impurity.

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Received on 30.12.2020 Revised on 10.02.2021

Asian Journal of Pharmaceutical Analysis. 2021; 11(2):159-169.

DOI: 10.52711/2231-5675.2021.00028

Groups	Description
Group 1	Carcinogenic to humans
Group 2A	Probably carcinogenic to humans*
Group 2B	Possibly carcinogenic to humans*
Group 3	Not classifiable as to its carcinogenicity to humans
Group 4	Probably not carcinogenic to humans

Class	Definition
1	Known mutagenic carcinogens
2	Known mutagens with unknown carcinogenic potential (bacterial mutagenicity positive*, no rodent carcinogenicity data)
3	Alerting structure, unrelated to the structure of the drug substance; no mutagenicity data
4	Alerting structure, same alert in drug substance or compounds related to the drug substance (e.g., process intermediates) which have been tested and are nonmutagenic
5	No structural alerts, or alerting structure with sufficient data to demonstrate lack of mutagenicity or carcinogenicity

Sr. No.	Name of impurity	Class of impurity	Impurity found in drug	Detection method
1.	NDMA (N-nitroso dimethylamine)	Nitrosamine	Ranitidine, Valsartan, metformin	GC-MS/MS, LC-MS, LC-HRMS, Rapid fire MS/MS, HPLC
2.	NDEA(N-nitrosodiethylamine)	Nitrosamine	Valsartan, irbesartan	GC-MS/MS, LC-HRMS, Rapid fire MS/MS
3.	NMBA (N-nitroso-N-methyl-4-aminobutyric acid)	Nitrosamine	Losartan potassium	LC-HRMS, Rapid fire MS/MS
4.	MNP (1-methyl-4-nitrosopiperazine)	Nitrosamine	Rifampin	LC-ESI-HRMS method
5.	CPNP (1-cyclopentyl-4-nitrosopiperazine)	Nitrosamine	Rifapentine	LC-ESI-HRMS method
6.	DMF (Dimethylformamide)	Solvent	Valsartan	GC-MS
7.	EMS (Ethyl methanesulfonate)	Sulfonate ester	Viracept	GC-MS