INTRODUCTION:

Nitroso compounds were first discovered by Adolf von Baeyer in 1863. In 1956, Barnes and Magee found that dimethylnitrosamine causes liver tumors in rats. N-nitroso compounds are classified into N-nitrosamines and N-nitroso amides (Fig. 1). Nitrosamines are stable compounds formed by a reaction between secondary or tertiary amines and nitrous acid under specific temperature and pH conditions¹.

Primary amines, however, do not form nitrosamines, as they produce reactive diazonium ions. N-nitroso amides are unstable due to the presence of both nitroso and carbonyl groups². N-nitroso compounds (NOC) can cause the human cancer by exposing in both endogenous and exogenous.


N-nitrosamide	N-nitrosamine

Figure 1: Structures of N-Nitros compounds

Carcinogenicity of Nitrosamine impurities:

Nitrosamines (NA) are not directly carcinogenic but can be converted into alkylating agents through metabolic activation, leading to DNA mutations³. The P450 enzyme, primarily found in the liver, plays a key role in this process. It catalyzes oxidation reactions, aiding in the metabolism of medicines, toxins, and biological substances. This oxidation activates nitrosamines, turning them into alkylating agents capable of modifying DNA and causing mutations.

Endogenous and Exogenous Nitrosamine formation:

NA exposure can be endogenous or exogenous. Endogenously, nitrosamine impurities can form in the stomach. Studies show that feeding animals nitrite and secondary amines leads to tumor formation, suggesting endogenous NA formation. In food, NA can form when a nitrosating agent, like nitrogen oxides, reacts with amines. Two major sources of nitrogen oxides in food are nitrate/nitrite addition and heating/drying during food processing. NA are found in high concentrations in dried meat, cheese, pickled foods, and food stored or processed under moist conditions⁴.

Recently, nitrosamine impurities were unexpectedly found in some human medicines. In July 2018, Prinston Pharmaceutical Inc. recalled Valsartan tablets due to trace amounts of N-Nitrosodimethylamine (NDMA) in the Valsartan API (Fig. 2) made by Zhejiang Huahai Pharmaceutical Co. Ltd.⁵. The U.S. EPA considers NDMA a probable human carcinogen based on animal studies. The acceptable intake is 96 nanograms of NDMA per day, deemed reasonably safe for humans.

In October 2018, ScieGen recalled certain Irbesartan products due to N-Nitrosodiethylamine (NDEA) contamination, a suspected human carcinogen. Irbesartan was the first non-Valsartan product found with NDEA impurity, and Aurobindo manufactured the API for ScieGen's Irbesartan (Fig. 2)⁶.

In November 2018, Sandoz voluntarily recalled Losartan tablets due to N-nitrosodiethylamine (NDEA) contamination, with the API manufactured by Zhejiang Huahai (Fig. 2)⁷. In March 2019, Losartan was again recalled, this time by Torrent, due to a new impurity, N-nitroso-N-methyl-4-aminobutyric acid (NMBA), in the API from Hetero Labs⁸. The common feature in Valsartan, Irbesartan, and Losartan is the tetrazole ring in their structure, which is also present in two other sartans, Candesartan and Olmesartan. The structures of all these 5 molecules are shown in Figure 2.


Valsartan	Irbesartan

Losartan	Candesartan

Olmesartan

Figure 2 Tetrazole ring containing API’s

To better control nitrosamines in APIs, the European Directorate for the Quality of Medicines and Healthcare (EDQM) revised Sartan monographs in the 10^th edition of Ph. Eur., adding interim limits. Unlike tetrazole-containing sartans, azilsartan, eprosartan, and telmisartan do not carry the same nitrosamine contamination risk⁹. The concern extended beyond sartans when NDMA was found as a contaminant in Ranitidine (Fig. 3) in September 2019¹⁰, Metformin (Fig. 3) in December 2019¹¹, and later in Nizatidine¹² (Fig. 3).


Metformin	Nizatidine

Ranitidine

Figure 3 Nitrosamine contaminated drug substances

Toxicity and acceptable intakes of Nitrosamine Impurities:

In various animal species NA compounds are identified as potential genotoxic nature are classified as possible human carcinogenic by the International Agency for Research on Cancer (IARC)¹³. ICH society NA compounds are classified as “cohort of concern”, and few guidelines are issued for industry are ICH M7. The acceptable intakes proposed by several health agencies are provided in the Table 1 along with the structure, IARC group and mutagenicity of respective nitrosamine compounds.

Table 1: Acceptable intakes of various Nitrosamine compounds

Nitrosamine compound name	Abbreviation	Structure	IARC group^@	Acceptable intake (AI) (ng/day)
Nitrosamine compound name	Abbreviation	Structure	IARC group^@	^#EMA	^#FDA	^#Health Canada	^#ANVISA
N-Nitrosodimethylamine	NDMA		2A	96.0	96.0	96.0	96.0
N-Nitrosodiethylamine	NDEA		2A	26.5	26.5	26.5	26.5
N-Nitroso-di-n-butylamine	NDBA		2B	26.5	-	26.5	26.5
N-nitrosomorpholine	NMOR		2B	127.0	-	-	-
N-nitrosodipropylamine	NDPA		2B	26.5	-	-	-
N-Nitroso ethylisopropylamine	EIPNA		-	26.5	26.5	26.5	26.5
N-Nitroso diisopropylamine	DIPNA		-	26.5	26.5	26.5	26.5
N-Nitroso-N-methyl-4-aminobutyric acid	NMBA		-	96.0	96.0	96.0	96.0
1-Methyl-4-nitrosopiperazine	MeNP/ MNP		-	26.5	-	96.0	26.5
N-Nitroso-N-methylaniline	NMPA		-	34.3	26.5	-	34.3
N-nitroso-varenicline	NNV		-	37.0	-	-	-

^@IARC Group – 2A: Probably carcinogenic to humans,IARC Group – 2B: Possibly carcinogenic to humans

# EMA - European Medicines Agency

FDA - Food and Drug Administration

ANVISA - Agência Nacional de Vigilância Sanitária (Brazilian Health Regulatory Agency)

Limit Calculation:

Nitrosamine’s limit can be calculated in two ways.

1. N-nitrosamines should be subjected to a substance-specific limit derivation process, in accordance with ICH M7, if adequate substance-specific animal carcinogenicity data are available to compute a dependable TD50.

2. The default option for N-nitrosamines can be a class-specific TTC of 18 ng/day (derived from the Lhasa carcinogenic potency database) if there is insufficient substance-specific data to derive a substance-specific limit for lifetime exposure, as advised by the ICH M7 guideline.

If properly justified, a method based on SAR concerns to determine an acceptable intake limit is acceptable.

Nitrosamine Contamination sources in pharmaceutical products:

Several root causes are identified for the formation of nitrosamines in pharmaceutical products. The details are given in Figure 4.

Figure 4 Contamination sources of nitrosamines

Controls of Nitrosamines:

Handle the risk with mutagenic impurities in a drug by using ICH M7 guidance. Total Four control options (refer to ICH M7; Section 8) are identified to regulate the mutagenic impurities. Out of four options, option 1 and 2 only testing is involved and option 3 and 4 are a combination of testing and experts’ knowledge.

Option 1: Impurity can be controlled in the API if the specification limit ≤ the acceptable limit.

Option 2: Impurity control in the starting substances / active intermediates control specifications with a specification limit ≤ the acceptable limit.

Option 3: Impurity control in the specification of a raw material/ starting material/ intermediate/ in-process control, with a specification limit > the acceptable limit and prove the process capability by spike-purge studies to guarantee the impurity level in the drug substance is below compound specific limit.

Option 4: Justification of control approach based on scientific principles.

Quantitative evaluation of Nitrosamines in Pharmaceutical products:

Quantitative evaluation of nitrosamines is challenging due to the low specification limits in tetrazole-containing sartans and other contaminated drugs. These limits are based on low acceptable intake values, drug dosage, and long-term use. Regulatory bodies in the US and EU require testing of at-risk medicinal products by September 26, 2022 (EU), and September 1, 2023 (US), three years after the guidance was published.

To test nitrosamine impurities in medicinal products, analytical methods must be developed and validated for specificity, precision, accuracy, and linearity. Regulatory agencies, research bodies, and the pharmaceutical industry have developed various methods using gas chromatography (GC-MS/MS), liquid chromatography (LC-MS/MS), and high-performance liquid chromatography (HPLC) with UV detection to meet current regulatory requirements. Few authors are wrote some reviews and research on Nitrosamine impurities in recent years shows its significance^14-28.

Purging of nitrosamines based on theoretical knowledge:

The purging of nitrosamine impurities is validated on theoretical thought of their physicochemical properties graphical representation was shown in Figure 5 and the detailed approach developed by Teasdale et al.,²⁹. Each physicochemical property has its own purge value are shown in Table 2.

Table 2 Standard Purge factors and their values

Influencing factor	Relative Purge scale	Inference
Rate of Reactivity	100	More reactive
	10	Reasonably reactive
	01	Least reactive
Relative solubility	10	Easily miscible
	3	Moderative soluble
	1	Partially miscible
Strength of vaporization	10	boiling point >20 °C
	3	boiling point within ±20 °C
	1	boiling point <20 °C
Rate of Ionization	10 - 3	genotoxic impurities are have different ionization potential rather than desired product treated
Chromatogram	10 - 100	Extent of Polarity
scavenger resins	3 - 100	Validated on individual criteria

The Purge score 100 shows 99% of the impurity removed.

Figure 5: Physicochemical parameters to calculate the purge value

Purge ratio:

Purge ratio =

Purge factor for the impurity

Required purge factor @ TTC or PDE

Based on purge ratio, analytical data evidential requirements are made and shown in Table 3.

Table 3 Evidential requirements based on purge ratio

Purge Ratio (PR)	Evidential Requirements
>1000	Option 4 supporting; can go with purge ratio
1000 < PR > 100	- Purge calculation - Literature evidence - Analytical data to support purge studies
100 < PR > 1	Option 4 supporting only with purge ratio - Purge calculation - Literature evidence - Analytical data to support purge studies
PR < 1	Option 4 not supported

CONCLUSIONS:

To cater current regulatory requirements and to ensure the quality of drug and patient safety; pharmaceutical industry should take utmost care towards new product developments and process optimization of the existing developed and marketed products to reduce the level of nitrosamines by implementing precautionary measures as discussed above. If at all nitrosamines present in the Active Pharma Ingredient substance, suitable analytical method to be developed and validated adequately to control the nitrosamines.

CONFLICT OF INTEREST:

There is no conflict of interest among the authors to publish this article.

AUTHORS’ CONTRIBUTIONS STATEMENT:

All the authors are collectively worked to do this research work at various stages of data collection, design of the work, data analysis, interpretation and drafting the article.

ACKNOWLEDGMENTS:

The authors are acknowledged to GITAM Central Research Laboratories, GITAM Deemed to be University, Visakhapatnam for providing necessary facilities to perform this research work.

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Received on 02.04.2024 Revised on 06.11.2024

Accepted on 08.03.2025 Published on 01.10.2025

Available online from October 04, 2025

Research J. Pharmacy and Technology. 2025;18(10):5041-5046.

DOI: 10.52711/0974-360X.2025.00728

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