Carcinogens refer to substances that can induce cancer in humans and experimental animals under certain conditions. Common carcinogens in drugs are nitrosamines, halogenated hydrocarbons, sulfonates and benzenesulfonates^[1]. Nitrosamines are a group of organic compounds containing nitroso functional groups (R₁R₂-N-NO), which often exist in trace concentrations in food, tobacco, sterilized drinking water, and plastic products^[2][3][4][5]. In recent years, domestic and foreign drug regulatory agencies have issued announcements pointing out that high levels of N-nitrosodimethylamine (NDMA) have been detected in some commonly used drugs (such as valsartan, ranitidine, nizatidine, metformin), which has aroused widespread concern among researchers and the public^[6].

In 2018, trace amounts of NDMA impurities were first detected in valsartan bulk drug exported by a Chinese manufacturer, which was reported to the National Medical Products Administration of China (CNMPA) on July 6^[7]. On July 29, CNMPA issued an announcement on the Valsartan API incident, stating that after the manufacturer involved detected NDMA impurities, it followed the relevant regulations and requirements.It took the initiative to disclose to the public the information about the batches of drugs involved in the incident and the concentration of impurities detected, and initiated voluntary recall measures, and immediately suspended the release and delivery of all valsartan raw materials in domestic and foreign markets^[8]. The manufacturer's current API process was approved by the European Medicines Agency (European Medicines Agency, EMA) and the Food and Drug Administration (U. S. Food and Drug Administration, U. S. FDA) in 2012 and 2013, respectively^[9]. As of July 23, the company has completed the recall of all domestic APIs^[10]. The EMA subsequently initiated a review of all valsartan products in the European Union, and found N-nitrosodiethylamine (NDEA) and N-nitro-N-methyl-4-aminobutyric acid (NMBA) impurities in other valsartan products^[11].

Since then, EMA has carried out Europe-wide testing of all human medicines that may present a risk of nitrosamines. In September 2019, EMA and the U. S. FDA jointly announced that high levels of nitrosamine harmful impurities were also detected in ranitidine, a commonly used gastric drug, with the highest level of NDMA reaching 860 ng per tablet^[12]. Based on the usual dosage, it is 9 times higher than the acceptable daily intake limit (96 ng/d) required by the U. S. FDA^[13]. Shortly after, NDMA was detected in another H2 receptor antagonist, nizatidine^[14].

In 2019, the U. S. FDA detected the content of NDMA ranging from 60 to 190 ng/tablet in some batches of finished metformin, indicating that the finished metformin may also be contaminated by NDMA^[15]. In April 2020, the U. S. FDA called for the immediate discontinuation of all ranitidine products as the safety evaluation of the drug progressed. The reason is that NDMA levels in ranitidine drug products may increase when stored at high temperatures, and can occur under packaging, handling, and even normal storage conditions^[16]. Currently, the U. S. FDA has set the acceptable maximum daily intake of valsartan for NDMA and NDEA at 96 ng/d and 26.5 ng/d, respectively^[17].

Valsartan, ranitidine and metformin are important drugs for the treatment of hypertension, gastric ulcer and diabetes, respectively. According to the CDC database, the United States issued 83.4 million prescriptions for hypertension, 25.2 million for gastric ulcer and 83.8 million for type 2 diabetes in 2018^[18][19][20]. Therefore, the detection of nitrosamine impurities in the above commonly used drugs may pose a threat to public health.

In the 1970s, researchers began to detect N-nitrosamine impurities in drugs^[21]. At present, the detection methods mainly include high performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS), high performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS), high performance liquid chromatography with ultraviolet detector (HPLC-UV), gas chromatography-mass spectrometry (GC-MS), headspace gas chromatograph-mass spectrometry. Since the "Valsartan Incident" in 2018, drug administrations in various countries have begun to pay attention to the detection of N-nitrosamine impurities in drugs. the detection methods issued by CNMPA, U. S. FDA, and European Agency for the Quality of Medicines (EuropeanDirectorate for the Quality of Medicines, EDQM) and the types of nitrosamines detected are shown in Table 1^{[22⇓⇓~25]}.

N-nitrosamines generally have good volatility and high polarity. Based on the separation mechanism of similarity and compatibility, high polarity liquid chromatography or medium polarity gas chromatography columns can be used for separation. The injection detection methods in gas chromatography mainly include headspace injection and direct injection. When N-nitrosamines are analyzed by headspace injection, the commonly used solvents are N-methylpyrrolidone (NMP), dimethyl sulfoxide (DMSO), etc. For direct injection, the common solvents are dichloromethane (DCM), acetonitrile (CAN), methanol (MeOH), etc.

In order to avoid the increase of nitrosamine level caused by the degradation of the tested substance at high temperature when gas chromatography is used for detection, ranitidine and other drugs that are easily degraded at high temperature can be detected by liquid chromatography. Liquid chromatography has become a more important method for the detection of nitrosamines in drugs, usually combined with mass spectrometry. The detection methods and concentration levels of nitrosamines in drugs are shown in Table 2.

After the "valsartan" incident, three research teams in Denmark, Germany and France carried out cohort studies^[60][61][62]. The findings are basically consistent, and exposure to valsartan containing NDMA is not associated with overall cancer risk, but it increases the risk of liver cancer, colorectal cancer, uterine cancer and melanoma.

On September 9, 2018, the University of Southern Denmark led a rapid publication of a cohort study including 5150 people^[60]. People with no history of cancer, aged 40 years or older, who were on valsartan on January 1, 2012, or who started valsartan from February 1, 2012 to June 30, 2017, were added to the cohort. Participants were followed from one year after entering the cohort (lag time period) until experiencing cancer outcome, death, migration, or the end of the study period (June 30, 2018). Median follow-up was 4.6 years. There were 104 cases of cancer in patients not taking nitrosamine-containing drugs and 198 cases of cancer in patients taking nitrosamine-containing drugs, with an adjusted hazard ratio for overall cancer of 1.09 (95% confidence interval: 0.85 to 1.41) and no evidence of a dose-response relationship (P = 0.70). For single cancer outcomes, there was an increased risk of colorectal cancer (hazard ratio 1.46, 95% confidence interval: 0.79 to 2.73) and uterine cancer (1.81, 95% confidence interval: 0.55 to 5.90).

In 2021, the German Center for Neurodegenerative Diseases led the publication of a cohort study involving 780,000 people^[61]. This cohort study was based on data obtained from a large German health insurance company. A total of 780,871 patients who were prescribed valsartan between 2012 and 2017 were included in the study. The study found no association between exposure to valsartan containing NDMA and overall cancer risk. However, a statistically significant association was found between exposure to valsartan with NDMA and liver cancer (aHR = 1.16, 95% confidence interval: 1.03; 1.31). Due to the current relatively short follow-up, the long-term effects of regular use of valsartan possibly contaminated with NDMA for more than 3 years cannot be assessed, and close observation of the potential long-term effects of valsartan contaminated with NDMA is recommended.

In 2022, the French National Agency for Medicines and Healthcare Products led the publication of the results of a large-scale cohort study involving 1.4 million people^[62]. The study was based on the National Health Data System, which includes health-related costs for all French residents, and targeted 1.4 million patients without a history of cancer who took valsartan between January 1, 2013 and December 31, 2017, aged 40 to 80 years. 986,126 and 670,388 patients were exposed to NDMA-contaminated and uncontaminated valsartan, respectively. NDMA-contaminated valsartan did not increase the overall risk of cancer (aHR = 0.99, 95% CI: 0.98 to 1.0). However, the risk of liver cancer (aHR = 1.12, 95% confidence interval: 1.04 to 1.22) and melanoma (aHR = 1.10, 95% confidence interval: 1.03 to 1.18) was higher in exposed patients. The study estimated an average of 3.7 and 5.8 additional cases of liver cancer and melanoma, respectively, per 100,000 people per year.

In general, nitrosamines are products between a nitrosating agent and a secondary or tertiary amine, i.e., the secondary or tertiary amine reacts with the nitrosating agent to form an N-nitrosamine. As far as we know, there are many reasons for nitrosamine impurities in drugs, such as process production, degradation pathway and pollution introduction.

It is pointed out in the Technical Guidelines for the Research of Nitrosamine Impurities in Chemical Drugs issued by China in 2020 that nitrosamine impurities may be introduced through the following ways: ① nitrosamine impurities are introduced by the process, such as the use of materials that can introduce secondary amines and nitrosation reagents in the same process step^[63]; ② Introduced by pollution, such as the use of materials contaminated by nitrosamine impurities in the production process; ③ Degradation, for example, ranitidine will produce nitrosamine impurities at high temperature.

It is reported that in the "valsartan incident", in order to increase the production of valsartan and reduce the waste of chemical agents, the manufacturer changed the synthesis process of tetrazole ring formation of valsartan pharmacodynamic substance, replaced tributyltin azide with anhydrous sodium azide, and used dimethylformamide (DMF) as solvent. In the production process, sodium nitrite forms nitrous acid in acidic medium, which nitrosates with dimethylamine (DMA) impurities in dimethylformamide to form NDMA (Fig. 2).

The source of nitrosamine impurities in drugs is also related to drug packaging and drug storage conditions. In its recall announcement on ranitidine (US market), the U. S. FDA revealed that if ranitidine drug substance is stored for a long time or at a high temperature, it will lead to an increase in NDMA impurities in the drug substance, and it is unacceptable for consumers to be exposed to nitrosamine impurities at this level for a long time^[16]. If ranitidine tablets were stored at 40 ℃ and 75% humidity for about eight weeks, the NDMA content in the tablets increased significantly from about 0.19 μg/G to 116 μg/G, which is much higher than the acceptable daily intake limit (0.32 μg/G)^[65]. The packaging of the drug, especially the cover foil, contains cellulose nitrate as a primer, as well as secondary amines commonly found in printing inks, which can produce trace amounts of N-nitrosamines in the cover foil, which in turn can contaminate the drug inside the packaging^[66].

In addition to the exogenous contamination of NDMA in the above drugs, some drugs containing secondary amine structures will react in vivo to form NDMA, resulting in endogenous contamination. Under simulated gastric juice conditions, the amount of NDMA produced by ranitidine increased with the increase of nitrite concentration and the decrease of pH value, reaching a level exceeding the standard by three orders of magnitude^[67]. The main evidence for the possibility of in vivo conversion of ranitidine to NDMA comes from a 10-person study^[68]. The study reported an approximately 400-fold increase in total urinary NDMA excretion from 110 ng to 47,600 ng and a more than 5-fold increase in total N-nitrosamine content to 139,000 ng in volunteers within 24 H before and after oral administration of ranitidine (150 mg). It should be pointed out that this study used gas chromatography-mass spectrometry for NDMA measurement, and there is a possibility that ranitidine decomposes to release NDMA at the time of detection, resulting in high measurement results^[69].

However, some studies have shown that ranitidine does not convert to NDMA in the human stomach^[70,71]. Lior et al. Added ranitidine (150 mg) to simulated gastric juice with different nitrite concentrations (6.9 ~ 690 mg/L), and the content of NDMA was not detected until the nitrite concentration reached 345 mg/L, suggesting that ranitidine would not be converted into NDMA in gastric juice under normal conditions. NDMA levels were also not elevated in simulated gastric and intestinal fluid tests at FDA facilities^[72].

To sum up, it is still a problem to be further studied how many nitrosamines exist in the human body after taking drugs from exogenous contamination and how many from endogenous generation.

As a new class of pollutants, nitrosamines have been widely concerned by researchers at home and abroad in recent years. Because of its strong carcinogenicity and high health risk, many countries and organizations have made clear regulations on its concentration limits in food and drinking water, among which NDMA is the most concerned one. The limits of nitrosamines in foods and beverages in different countries are shown in Table 5.

For food, Russia and Ukraine have formulated the most stringent standards for nitrosamines, Iceland is more relaxed, and other countries have not yet formulated the limit standards for nitrosamines in food. China has stipulated that the NDEA limits of meat products and seafood are 5 μg/kg and 7 μg/kg, but the current standards only retain the NDMA limits of 3 μg/kg and 4 μg/kg in meat products and seafood. For beer, most Western European countries set a content limit of 0.5 μg/kg, while the United States and Japan have the most relaxed standard of 5 μg/kg. China has set a content limit of 3 μg/kg for NDMA in beer in the Hygienic Standard for Fermented Wine (GB2758-81, which has been abolished). However, the current National Food Safety Standard for Fermented Wine and Its Blended Wine (GB2758-2012) cancels the requirement for NDMA in beer^[73].

The limit of nitrosamines in drinking water is much stricter than that in food, because drinking water is the largest and most important livelihood product, which is related to all aspects of people's daily life. At present, Ontario, Canada stipulates that the limit of NDMA in drinking water is 9 ng/L, California (10 ng/L), Massachusetts (10 ng/L) and Canada (40 ng/I) have also formulated the standard of nitrosamines in drinking water, and the World Health Organization (WHO) has set the limit of NDMA in drinking water as 100 ng/I. Some cities in China, such as Shanghai, Shenzhen, Zhangjiakou and Suzhou, have formulated a standard of 100 ng/L for nitrosamines in drinking water according to the Guidelines for Drinking Water Quality of the World Health Organization. NDMA is listed in the appendix of the newly revised Sanitary Standard for Drinking Water (GB 5749-2022), and the recommended limit is also 100 ng/L.

In recent years, researchers have analyzed the health risks of nitrosamines ingested by Chinese residents through food, drinking water and tobacco. Li et al. Estimated the lifelong carcinogenic risk (food :1.74×10^-4, drinking water :1.38×10^-5) of residents in 20 provinces of China due to NDMA and NDEA in food and drinking water^[74]. Sang et al. Calculated that the average lifetime carcinogenic risk caused by NDMA in food for Chinese residents was 3.61×10^-5, and the average lifetime carcinogenic risk caused by NDMA in drinking water was 3.99×10^-6[75]. Other researchers have also reported the lifelong carcinogenic risk of water sources in some areas of the provinces and cities (counties) along the Yangtze River Basin in China, including Guan Yue (3.58×10^-6~1.42×10^-5), Roman (1.06×10^-5~3.87×10^-4), Luo Qiong (1.42×10^-5~3.39×10^-5), etc^[76][77][78]. Li et al. Estimated the lifelong carcinogenic risk (3.8×10^-4) of Chinese smokers due to nitrosamines (including tobacco-specific nitrosamines, NDMA, NDEA) in tobacco^[79].

The author used the carcinogenic risk caused by nitrosamines in drinking water (1.38×10^-5), food (1.74×10^-4) and tobacco (3.8×10^-4) estimated by Li et al., combined with the 75th percentile carcinogenic risk level (5.61×10^-4) caused by nitrosamine impurities in substandard drugs calculated in this paper.The carcinogenic risk caused by the four lifestyles of Chinese residents before the recall of substandard drugs was estimated, which were: food + drinking water (1.88×10^-4), food + drinking water + substandard drugs (7.49×10^-4), food + drink water + smoking (5.68×10^-4), and food + drink water + substandard drug + smoking (1.13×10^-3), as shown in Fig. 3.

It can be seen from Figure 3 that the carcinogenic risk caused by taking drugs containing nitrosamine impurities is 399% higher than that of healthy people. The carcinogenic risk of nitrosamines increased by smoking is 302% higher, and the carcinogenic risk caused by substandard drugs is even higher than that caused by smoking. When people who take drugs smoke at the same time, they may increase the risk of cancer by 601% compared with healthy people.

After the drug recall incident, China's Food and Drug Administration has strengthened the detection and supervision of drug impurities, and finished drug and API enterprises have also strengthened the detection of their own products, resulting in a significant reduction in the impurity content of drugs. As shown in Table 2, after 2020, many scholars in China have tested valsartan, irbesartan, candesartan, ranitidine, metformin and other drugs in China, and found that no raw materials and finished drugs exceeded the standard, and most of the results were below the detection limit.

In view of the potential safety hazards caused by genotoxic impurities to patients taking drugs, relevant drug regulatory agencies at home and abroad have successively issued a series of policy documents (Table 6), which have mainly gone through three stages of development: the initial regulatory gap, the pursuit of complete avoidance, and the reasonable reduction of the actual situation.

In the above documents, the regulatory agencies analyzed the impurities in the products, investigated the causes and formulated a number of standards and measures, the core issue of which is how to limit and control the content of genotoxic impurities. On the basis of the previous documents, EMA issued the draft (2004) and final (2006) guidelines on the limits of genotoxic impurities, and the concept of Threshold of Toxicological concern (TTC) was proposed for the first time, and the document suggested that the exposure of genotoxic impurities should be limited by TTC value^[80]. TTC refers to the exposure of genotoxic impurities corresponding to 50% tumor incidence (TD₅₀ value) by simple linear extrapolation to 1/100000 tumor incidence. The EMA sets the TTC value at 1.5 μg/d, which is 1.5 μg of mutagenic impurity per person per day, and the risk of tumor development is 1 in 100,000 in the case of lifelong intake (based on a life span of 70 years). However, the standard of TTC has some limitations. First, the TTC value is not suitable for patients with short medication cycles, which is too conservative. Second, the TTC value is difficult to apply to all genotoxic impurities.

in 2014, the International Coordinating Council (International Council for Harmonization, ICH) issued the M7 (R1) Assessment and Control ofDNA Reactive (Mutagenic) Impurities in Pharma ceuticals To Limit Potential Carcinogenic Risk Guidelines^[81]. The document was upgraded on the basis of the regulatory ideas of EMA and U. S. FDA, and the genotoxic impurities were classified into five categories by using the strategies of "segmented TTC" and "five-classification system", and the corresponding control strategies are shown in Table 7 and Table 8, which provided a reference for drug research and development under the environment at that time. At the same time, the document points out that when a drug product contains multiple category 1 impurities, such as nitrosamines, the acceptable intake of these highly effective carcinogens may be much lower than the acceptable intake specified in this guideline, and a case-by-case approach should be developed to control each impurity separately according to its specific limit. When the drug product contains two Class 2 or Class 3 impurities, and the acceptable intake of these impurities is close to the acceptable intake specified in this guideline, the control of each impurity is still controlled according to the principle of "piecewise TTC".

In the International Coordinating Council (ICH) M7 guideline, it is pointed out that N-nitrosamines are special genotoxic impurities with high carcinogenic activity, and the TTC method is not enough to control the risk of N-nitrosamines, which needs special attention. The maximum allowable daily intake of N-nitrosamines was calculated according to the linear extrapolation of the TD₅₀ value (which can be obtained by searching the CPDB database) to the carcinogenic risk of 1/100,000, and the results are shown in Table 9.

China joined the International Coordinating Council (ICH) in July 2017, and then gradually carried out and improved the research on harmful impurities in drugs. In 2019, the National Pharmacopoeia Committee issued the Guidelines for the Control of Genotoxic Impurities (draft for comments), including hazard assessment methods, acceptable intake calculation methods and limit formulation methods.The acceptable intake of impurities is shown in Table 9, which refers to the formulation method and standard in the M7 guideline of the International Coordinating Council (ICH), and the acceptable intake of impurities remains unchanged^[22]. In 2020, the Technical Guidelines for Nitrosamine Impurities in Chemical Drugs (Trial Implementation) published on the website of the State Drug Administration pointed out that nitrosamine impurities with very small limits can cause harm to human body, and as trace impurities, their detection and control are difficult.Therefore, the strategy of "avoidance first, control second" should be adopted for the monitoring of nitrosamine impurities. Avoidance first refers to the control from the source of drug production, the control of the process conditions and chemical agents of raw materials for drug production, and the avoidance of nitrosamine impurities as far as possible^[82]. Control as a supplement refers to the overall regulation of the production process steps, which advances or optimizes the production process and storage conditions of some unavoidable processes with the risk of nitrosamine impurities, so as to ensure that the nitrosamine impurities in finished products are below the limit requirements. For chronic administration, the guideline uses the TD₅₀ of the most sensitive target organ from the most sensitive sex and species to calculate the acceptable intake according to the International Coordinating Council (ICH) M7 guideline. If the TD₅₀ of NDMA in rats and mice are 0.0959 mg/kg/d and 0.189 mg/kg/d, respectively, and the body weight of an adult is based on 50 kg, and the dose is linearly extrapolated according to the calculation method in the M7 guideline of the International Council for Harmonization (ICH), the maximum acceptable daily intake of NDMA is 0.0959 mg/kg/d × 50 kg/50 000 = 96 ng/d. However, the guidelines do not mention regulatory limits for short-term administration. The purpose of the guidelines is to provide guidance for the development and monitoring of nitrosamine impurities in biochemical drugs registered for marketing or issued.

Since the discovery of nitrosamine impurities in valsartan in a domestic enterprise in 2018, more and more researchers and the public have paid attention to nitrosamines, and the control of nitrosamine impurities in drugs has also been highly valued by various regulatory agencies and manufacturers. Medicinal products are designed to improve the health of patients, so genotoxic and carcinogenic impurities above a certain level are unacceptable. Manufacturers and regulators need to work together to solve this problem, thoroughly solve the problem of NDMA formation in the manufacturing process, and establish more efficient and sensitive quantitative detection methods to ensure the quality and safety of available drugs.

In this paper, valsartan has the highest concentration of nitrosamine impurities (NDMA content is not detected ~ 20.19 μg/tablet, NDEA content is not detected ~ 1.31 μg/tablet), resulting in the highest additional carcinogenic risk, and the median value of 4.69×10^-6,75 percentile is as high as 5.61×10^-4, indicating that at least 25% of drugs have excessive carcinogenic risk. Nitrosamine impurities in ranitidine and metformin are much lower, and their carcinogenic risk is close to or below the safe level of 10^-6. The carcinogenic risk of nitrosamine impurities in unqualified drugs is much higher than that in food and drinking water, even higher than that in tobacco, which needs to be paid great attention to. After strengthening supervision, there are no reports of excessive nitrosamine impurities in raw materials and finished drugs in China after 2020.

At present, China has become a major producer of raw materials and a major consumer of finished drugs. Therefore, China's drug regulatory agencies and research teams should strengthen the research on the formation, control and toxicity evaluation of toxic impurities in drugs, find a set of standards system in line with China's specific national conditions as soon as possible, and establish more stringent and operable regulatory measures. At the same time, the food and drug industry has zero tolerance for NDMA impurities. We should establish a clear and effective response method for sudden NDMA impurity events on the basis of a comprehensive grasp of NDMA impurity events, so as to provide a reference for dealing with other emergencies in the future.