N-Nitrosamines in Meat Products: Formation, Detection and Regulatory Challenges

Abstract

Nitrosamines (NAs) are a class of chemical compounds predominantly formed during the processing, curing, and storage of meat products through the reaction of nitrites with amines. Decades of toxicological and epidemiological evidence have unequivocally established several NAs as potent human carcinogens, with strong associations with gastrointestinal, pancreatic, and liver cancers. This review critically examines the pathways of NA formation in meat, the influence of processing conditions, and the factors contributing to their variability in food products. It also outlines state-of-the-art analytical techniques for their detection and summarizes recent scientific efforts to reduce their formation. Despite scientific consensus on the health hazards posed by dietary exposure to NAs, regulatory control remains fragmented and insufficient. Therefore, this review highlights the pressing need for coordinated international action and the development of a harmonized regulatory framework to mitigate public health risks.

1. Introduction

In the human diet, meat plays a prominent role due to its attractive flavor and texture, and it also serves as a key source of protein, vitamins, calcium, iron and minerals with high nutritional value [1]. Meat products are highly perishable, which is the result of degradable processes that occur due to the action of microorganisms, the enzymatic activity of meat, and the influence of various external, physical or chemical factors [2]. Today, meeting consumer demand remains challenging for high-quality and sensory-appealing meat products without the use of food additives. Additives are now considered essential technological components in the industrial production of processed foods because they enable the preservation of quality, extension of shelf life and improvement of product safety. It is almost impossible to find food on the open market that does not contain at least some form of additive [3]. After adding different spices to fresh meat, it is processed in various ways such as high-temperature frying, curing, smoking, grilling and cooking to produce products such as sausage, ham, bacon, dried meat and canned meat. By processing meat, its quality, organoleptic properties, preservation and safety are improved [4].

Salt has been used since ancient times to store and preserve meat and other foods, until it became clear in the nineteenth century that nitrates in salt water act as inhibitors of microorganisms [5]. Currently, the principal additives that can ensure the microbiological safety of cured meat products are nitrites, nitrates after they are reduced to nitrites, and their potassium and sodium salts [6]. In addition, these additives play a key role in the preservation of flavor, red color stability and product safety, i.e., they prevent the development of rancidity, unpleasant smell and taste during storage. Nitrite is well known for its bacteriostatic and bactericidal action against pathogenic bacteria, including Salmonella enterica, and species of Listeria and Clostridium botulinum [7,8,9,10].

The safety of cured meat products has remained a subject of ongoing concern of numerous studies since the 1970s, because nitrites added as additives can react with secondary amines and thus form N-nitrosamines (NAs), many of which are known carcinogens [11]. Although many studies have reported the presence of NAs in environmental samples such as plants, mud, soil, air, water, cosmetics and tobacco, food is known to be the primary source of human exposure to NAs [12,13]. Cured meat products often contain measurable levels of NAs mainly due to the use of nitrite as a preservative and are affected by several additional factors such as temperature, pH, processing conditions and the presence of free amines, especially biogenic amines [8]. In addition to meat products, the occurrence of NAs has also been recorded in other foods, such as beer, dried fish, cheese, soy sauce and certain vegetables [14].

Considering the multifaceted pathways of NA formation, their diverse origins, and the serious health concerns associated with their presence in food, a comprehensive understanding of their behavior in meat products is essential. This review brings together current knowledge on their formation, detection, regulation and mitigation, offering a critical overview for scientists, regulators and food industry professionals alike.

2. Formation and Occurrence of NAs in Meat Products

Numerous complex chemical reactions that lead to NA formation, such as Maillard reactions and saccharification, occur during the production of meat products. Maillard reactions represent a typical thermal reaction that enables other chemical changes [15]. In addition, food additives and the physical composition of food also play an important role in the formation of NAs [16]. Regardless of the abundance of precursors that favorably influence the formation of NAs, some characteristics of meat products can hinder their formation, such as low water activity and insufficiently low pH [15]. The rate of synthesis of NAs is proportional to the square of the concentration of nitrites, so lowering the concentration of nitrites in meat products has a significant impact on reducing the amount of NAs [17].

In meat products, the interaction of secondary amines with nitrites at high temperatures and low pH generates the formation of NAs [18]. In addition, the amount of NAs formed in production mainly depends on the intake of nitrite, and its concentration increases under favorable pH conditions, long storage times, high temperatures [19], and the presence of catalysts or inhibitors of the reaction mechanism [20]. In theory, the optimum pH for the nitrosation reaction is from 3.0 to 4.0. A high pH is not favorable for the conversion of nitrogen oxides and results in a slow reaction. On the other hand, too low a pH value leads to the protonation of amino compounds, making interaction with nitrogen oxides difficult [21]. Shakil et al. [22] found that the acidification process during meat fermentation can catalyze the production of NAs. When exposed to a low pH environment, sodium nitrite in meat products is transformed into nitrous acid, an unstable molecule, which then converts to nitrous anhydride (N₂O₃). Nitrous anhydride during the nitrosation reaction together with secondary amines, forming NAs [23,24]. N₂O₃ is formed at high temperatures by the degradation of nitroso derivatives during fat oxidation [25]. It is important to note that primary amines are quickly degraded to alcohol and nitrogen, while tertiary amines do not produce nitrites [26]. N₂O₃ is also formed in acidic conditions from nitrite (NO⁻²), which is then converted into NO and NO₂. Unprotonated secondary amines can react during the process of nucleophilic substitution with NO⁻, which is produced in the presence of a transition metal ion like Fe³⁺, to form NAs [18].

Factors such as cooking temperature and method, residual nitrite levels, concentrations of NA precursors and inhibitors, and storage conditions influence the concentration of NAs in meat products [27]. In research conducted by Sallan et al. [28], heat-treated sucuk sausage produced from beef meat and fat was cooked in different time periods of 1, 3, 5 and 7 min, and it was found that increasing the cooking time also increased the concentration of certain NAs, NDMA, NPYR and NPIP. This study demonstrated the influence of processing time on the formation of NAs, while another study by Drabik-Markiewicz et al. [29] demonstrated the influence of temperature on their formation. According to this study, in meat heat-treated above 120 °C, there was an increase in NDMA, and an increase in NPYR at temperatures above 200 °C. Another study showed that cooking methods with temperatures above 130 °C increase the risk of NA formation, with frying and grilling processes being particularly prominent [25]. In their research, Yurchenko and Mölder [23] also prove that temperature increases during the process of cooking result in a greater amount of NAs. Regarding the heating method, Li et al. [8] concluded that deep frying and pan frying promote the formation of high content of NDMA, NDEA and NPYR, whereas boiling and microwave treatment do not induce the production of NAs in sausages, indicating that these are safer methods from a food safety perspective. Furthermore, Lee et al. [30] observed that direct heating using charcoal, electric, or gas stoves produced more NDMA than using electric and microwave ovens and steam pans.

The content and type of protein greatly affect the formation of NAs, and according to one study, a slight increase in NDMA and NPYR in pork sausages was demonstrated when the fat content increased from 12 to 25% [27]. According to research by Drabik-Markiewicz et al. [31], the effect of the amino acids proline and hydroxyproline on the formation of NPYR was proven, while the effect of proline on the formation of NDMA did not exist, in contrast to the mild effect of hydroxyproline. Another amino acid, lysine, is a precursor to NPIP [15]. Furthermore, De Mey et al. [15] proved in their research that glycine, lysine, methionine, carnitine, choline and lecithin can form dimethylamine, which results in the production of the NDMA. Other works also confirm that amino acids such as glycine, sarcosine, L-alanine and L-valine mostly form NDMA, L-alanine is a precursor for the formation of NDMA and NDEA and in smaller amounts of NMEA, while L-proline can form NPYR [21].

High concentrations of NAs have been recorded at a high rate in bacon, sausages and ham, while unprocessed meat showed low or no NAs. NA content changes depending on cooking methods, temperature, time, food moisture or fat composition [8,12].

Table 1. Reported concentrations of detected NAs in various meat products across selected studies. N = number of samples analyzed; ND = not detected. LOD varied between studies depending on the analytical method used (e.g., GC-MS/MS, LC-MS/MS) and are reported as described by the original authors.

Meat Products	Concentration (µg/kg)						Literature
	N 1	NDMA	NDEA	NPYR	NPIP	NDBA
Bacon, uncooked	23	-	-	-	-	-	[32]
Chinese pork sausage	1	3.2	1.1	0.7	1.1	1.4	[33]
Chinese cured pork	1	2.4	ND	ND	1.4	0.6	[33]
Bacon	1	1.4	0.4	0.3	0.4	1.7	[33]
Bacon smoked	23	0.3–2.7	ND–1.0	ND–7.9	ND–0.1	ND–0.2	[34,35]
Bacon fried	365	ND 2–5.0	ND–0.7	ND–200.0	ND–1.2	ND–0.3	[23,32,36,37,38,39,40,41,42,43,44]
Bacon fat fried	65	4.0–20.2	–	15.8–32.0	ND–1.5	–	[36,41,45]
Bacon, cooked in microwave	24	0.3–4.5	-	0.1–21.3	-	-	[42,46]
Beef sausage	6	0.3	0.3	0.6	1.1	0.4	[47]
Pork, smoked	1	<0.8	<0.6	0.7–0.8	1.5–2.4	0.3–06	[48]
Black pudding	4	3.5	-	2.1	2.0	3.4	[49]
Chorizo sausages	10	ND–109.4	ND–0.4	ND–0.9	ND–0.001	-	[46,50]
Frankfurter sausage	24	0.2–2.2	-	ND–0.5	ND–1.9	-	[23,46,49,51]
Hot dog	1	2.1	ND	ND	0.2	0.4	[33]
Ham, cooked	108	0.2–4.9	ND–2.7	ND–5.3	ND–1.8	ND–4.6	[23,46,48,49,51,52]
Ham	21	0.6	ND	ND	0.2	0.2	[33]
Ham, smoked	42	0.1–3.1	ND–1.5	ND–19.5	ND–4.5	ND–2.6	[23,35,38,49,53]
Ham, Turkey	4	3.8	-	2.8	1.2	-	[49]
Chicken salami	4	2.4	ND–2.3	2.3–3.5	2.4–2.6	1.0–4.0	[54]
Beef salami	22	0.1–3.6	ND–3.6	ND–4.6	0.5–4.4	0.3–4.2	[54]
Turkey salami	6	0.2–3.2	0.3–3.9	ND–4.0	0.3–6.0	1.6–2.0	[54]
Liver pâté	13	0.3–2.9	ND–1.9	ND–1.4	0.1–0.9	ND–0.3	[35,49]
Mutton (or lamb), uncooked	9	-	-	-	-	-	[23]
Mutton (or lamb) fried	10	1.0	0.6	2.6	-	0.3	[23]
Canned pork	22	1.1–3.3	ND–62.8	ND–2.4	1.0–1.6	ND–55.6	[23,49,55]
Tinned meat	30	0.3–2.7	-	-	0.5–1.1	-	[56]
Cooked pork, seasoned	17	0.8–2.0	ND–0.5	ND–11.3	ND–1.6	ND–0.2	[23,38,51]
Pork fat-free, fried	10	0.4–0.6	0.2–0.3	2.4–3.6	0.3–0.5	0.2–0.4	[23]
Pork fat-fried	10	3.2–4.9	0.5–0.8	14.1–24.4	1.0–1.6	0.4–0.7	[23]
Pork, barbecue	9	ND–1.2	ND–0.3	ND–6.5	ND–1.5	ND–0.2	[23,40]
Pork, pickled	91	0.8	-	0.4	0.3	0.2	[23]
Pork, smoked	55	0.1–1.3	ND–1.6	ND–7.5	ND–2.9	ND–0.7	[23,35,38]
Pork, uncooked	8	-	-	-	-	-	[23]
Pork sausages	13	ND–3.3	-	ND–0.4	ND–0.2	-	[49,51]
Poultry, canned	13	0.93–0.94	ND–3.0	ND–2.0	ND–1.1	0.5–68.4	[23,55]
Poultry, fried	23	1.2–1.3	0.7–0.9	15.2–20.7	1.1–1.1	0.3–0.4	[23]
Poultry, cooked or grilled	20	0.9–5.0	ND–20.0	ND–8.4	ND–1.7	ND–0.2	[23,32,44]
Poultry, grilled, seasoned	10	1.4	0.6	14.6	2.0	0.3	[23]
Poultry, smoked	14	0.7–2.1	ND–0.3	0.4–22.1	0.3–5.3	0.2–6.3	[23,53]
Poultry, uncooked	10	-	-	-	-	-	[23,32]
Poultry, other products	4	ND–0.5	ND–0.8	ND–0.4	ND–2.6	ND–2.8	[35,38,49]
Salami	148	ND–5.0	ND–4.6	ND–2.7	ND–1.2	ND–1.7	[23,35,38,47,49,51]
Sausage, cooked	54	ND–1.5	ND–3.0	ND–2.3	ND–1.8	ND–0.3	[23,32,35,44,47,48,51]
Sausage, fried, grilled	71	0.2–3.6	0.1–2.6	ND–6.66	ND–2.6	ND–3.3	[23,35,52,53]
Sausage, smoked	174	0.1–1.4	0.1–2.6	ND–3.1	ND–2.3	ND–0.6	[23,35,52,53]

¹ N—number of analyzed samples. ² ND—not detected (<LOD). Note: Detailed treatment parameters (e.g., processing conditions, storage duration, curing methods) were not included in Table 1 due to inconsistent or incomplete reporting across the cited studies. Readers are encouraged to consult the original publications for specific methodological details where available.

shows data related to the amount of NAs in meat and meat products in the USA and Europe.

3. Sources of Nitrites and Amines in Meat

By definition, processed meats are those that undergo smoking, curing, salting, fermentation, and other processes that aim to improve organoleptic properties and extend shelf life. A commonly applied conservation technique in the meat industry is the addition of nitrite and nitrate salts, a process generally referred to as curing [11].

Sodium and potassium nitrate and nitrite are preservative additives that are often used in meat products. In the case of cured meat products that are not thermally processed, these additives play a significant role in microbial growth and proliferation [57]. Nitrates do not have a certain technological function, except that they serve as a nitrite reservoir. Given that nitrates are progressively reduced to nitrites, this is particularly important in the production of products with a long curing period [20]. Adding nitrites in the production process of cured meat has a significant technological role in terms of maintaining sensory properties [24]. By using salt as a preservative for meat products, producers began using those salts that would generate a tempting color and a distinctive aroma, as opposed to a less appealing gray tint [58]. The most important sensory effects of this additive are the preservation of the pinkish-red color [59] and participation in the preservation of flavor by preventing lipid oxidation [18,60]. Sodium nitrite (NaNO₂) inhibits protein oxidation, and disrupts the chain reaction of free radicals and complexes metal ions to inhibit fat oxidation [61]. The most important role inherent to nitrites is certainly the maintenance of microbiological safety. Prevention of pathogenic multiplication and toxinogenesis, especially the dangerous bacteria Clostridium botulinum, is still a strong reason for further use of this additive in the meat industry [62,63,64].

Besides the beneficial effects in processing, the addition of these additives can result in increased levels of NAs [27]. NaNO₂ during the curing process and gaseous nitrogen oxide during the smoking process are the main sources of nitrosyl donors, which react with secondary amines to form NAs [65]. By adding nitrite in concentrations of 0, 50, 100 and 150 mg/kg to beef sausage, Sallan et al. [28] in their research proved the impact of increasing nitrite concentration with an increase in the concentration of NAs.

NAs are formed in a reaction of a nitrosation agent with secondary amines [66], the origin of which is usually the content of fat when it comes to meat products, amino acids (proline, ornithine and lysine), biogenic amines (putrescine and cadaverine), elevated temperature, piperidines from pepper, as well as precursors formed by the breakdown of lipids and proteins during aging [15,67]. Secondary amines, as important prerequisites for the formation of NAs, are formed by excessive oxidation of proteins as well as by cyclization and deamination reactions of biogenic amines that are components of meat. More precisely, proteins in meat can be degraded by endogenous enzymes, proteases such as cathepsin, calcium neutral protease, aminopeptidases and dipeptidases, and by microbial enzymes, resulting in secondary products, peptides, amino acids and biogenic amines [15,50]. Biogenic amines are low molecular weight substances with biological activity that are formed by the decarboxylation of amino acids or the amination and transamination of aldehydes and ketones during cellular metabolic processes [68]. Also, it is important to note that microorganisms act as reductants and reduce nitrates to nitrites. This contributes to protein degradation to form amines and amino acids [65]. The most common biogenic amines produced by food spoilage are cadaverine, putrescine, spermidine, spermine, β-phenylethylamine, tyramine, and histamine. Of these, spermidine and spermine are naturally present in fresh meat [50,69]. Along with curing salt, spices, herbs and some vegetables are also important sources of nitrosating substances. It is necessary to consider that despite marked meat products that do not contain nitrites, the use of plant powders in the production of such products that contain a high level of nitrates acts as an additional source of NAs [21].

4. Analytical Methods of Determination

Many NAs detected in food, such as NDMA, NDEA and NDPA, have been characterized by volatility, and therefore gas chromatography has extensively been used for this type of analytical determination. Regarding non-volatile NAs (NVNAs), some analytical approaches use liquid chromatography (LC) with different detectors, but both techniques are applied for the simultaneous determination of volatile (VNAs) and non-volatile NAs in food and other matrices, and several analytical methods for the determination of NAs in food products are available in the literature [70,71,72,73,74]. Today’s mass spectrometers provide greater specificity than previously used detectors, making tandem mass spectrometry (MS/MS) detection the most commonly used tool for this analysis.

Chromatographic separation of VNA compounds in processed meat has been achieved using mainly gas chromatography (GC) [23,49,74]. A method developed for the analysis of processed meat samples, which includes both VNAs and NVNAs, was described by Herrmann et al. [38] where the limit of quantification (LOQ) varied and was too high considering the occurrence for different meat products due to the complexity of the matrix and interference, therefore the robustness of the method was limited. Lehotay et al. [75] developed and validated a GC-MS/MS method with a sample preparation procedure based on the “quick, simple, inexpensive, efficient, robust and safe” (QuEChERS) approach. The reporting limit of this method is 0.1 ng/g. However, the scope of the method was quite narrow as only five VNAs were involved and the validation study included only bacon. Niklas et al. [11] developed a method for quantifying the analysis of seven VNAs and two NVNAs in a larger selection of processed meat products. The limit of quantification achieved was 0.1–0.5 ng/g for most VNAs, and 2.3–4.2 ng/g for NVNAs. An overview of the NAs in meat products and the limits of detection can be found in

Table 2. Modern analytical methods used to determine NAs in meat products.

Analytical Method	Analyzed NAs	LOD (µg/kg)	Type of Sample	Reference
GCxGC/NCD	NDMA, NDEA, NDBA, NPIP, NPYR, NDPA	1.61–3.86	meat product	[47]
GC/MS	NDMA, NMEA, NDEA, NDBA, NPIP, NPYR, NMOR, NDPA, NDPhA	0.003–0.014	meat product	[49]
GC/CI-MS	NDMA, NMEA, NDEA, NDBA, NPIP, NPYR, NDPA	0.01–0.12	meat product	[33]
GC/FID, GC/MS	NDEA, NPIP, NPYR, NMOR	0.47–1.48	meat product	[76]
LC-(APCI/ESI) MS/MS	NDMA, NMEA, NDEA, NDBA, NPIP, NPYR, NMOR, NDPA, NMA, NSAR, NPRO, NTCA, NMTCA	0.2–1.0	meat product	[38]
GC-CI/MS	NDMA, NMEA, NDEA, NDBA, NPIP, NPYR, NMOR, NDPA, NDPhA	0.15–0.37	meat product	[48]
GC-MS/MS	NDMA, NDEA, NDBA, NPIP, NPYR	0.1	meat product	[75]
GLC/MS	NDMA, NMEA, NDEA, NDBA, NPIP, NPYR, NDPA	0.1–0.5	meat product	[77]
GC-MS/MS	NDMA, NMEA, NDEA, NDBA, NPIP, NPYR, NMOR, NDPA	0.05–0.10	beef meat	[78]
HS-SPME-GC-MS	NDMA *, NMEA, NDEA, NDBA, NPIP, NPYR, NMOR, NDPA, NDPhA **	0.16–3.6 * 56 ** 16	meat product	[79]
HS-SPME-GC–MS	NDPA, NDEA, NMEA, NDPA, NDBA, NPIP, NPYR, NMOR, NDPhA	1.45–3.15	raw meat	[80]
HPLC/UV-DAD	NMEA, NDEA, NDBA, NPIP, NPYR, NMOR, NDPA, NDPheA, NMA, NDBzA	20.1–111.6	meat product	[74]
LC-(APCI/ESI) MS/MS	NDMA, NPRO, NTGA, NMEA, NPYR, NMTGA, NDEA, NPIP, NDPA, NDBA	0.1–4.2 ng/g (LOQ)	cured meat products	[11]

* LOD (µg/kg) for NDMA. ** LOD (µg/kg) for NDPhA.

.

5. Health Risks Associated with NAs

Exposure to food-related hazards encompasses ingestion of food chemicals such as contaminants, pesticides, or drugs that are unintentionally present or intentionally added to food for processing purposes. Chronic exposure occurs when a person is continuously exposed to substances such as acrylamide, mycotoxins, or NAs over a long period of time [65]. NAs are a group of carcinogens that cause acute toxicity and mutagenicity. They have been discovered in numerous dishes, especially those containing nitrites and nitrates like meat products, in various alcoholic beverages, and can also appear as occupational pollutants in the workplace [17]. In addition to concerns related to the elevated content of NAs in meat products and the significant share of meat in the human diet, it has been confirmed that NAs increase the risk of cancer in people who consume meat products [27]. In 1997, the conclusions of the World Cancer Research Fund showed that high consumption of processed meat is associated with an increased risk of stomach cancer [81]. NAs are formed in meat products simultaneously during production, and can also occur during storage. Consuming these products may result in the accumulation of NAs in the body and therefore pose a significant threat to human health [4].

In general, NAs can be divided into two categories: volatile and nonvolatile [65]. Volatile NAs have high carcinogenic potential, while non-volatile NAs have weak or no carcinogenic potential [20]. Many volatile NAs are potent mutagens, and their dietary intake can induce tumors in various organs. Approximately 200 different NAs have been shown to be carcinogenic in more than 30 animal species. Human exposure to these compounds undoubtedly makes them susceptible to their carcinogenic effects [82]. Numerous studies have demonstrated a correlation between the consumption of red and processed meat and the development of various forms of cancer, especially colorectal [83], stomach [81] and pancreatic cancer [84], but it can also lead to the development of cardiovascular and other deadly diseases [85]. According to research on various types of experimental animals, it is proven that NAs are linked to numerous diseases and are known to induce tumors of the liver, lung, esophagus, bladder and pancreas [4].

Numerous negative effects of oral intake of NAs into the body through food, especially their carcinogenicity, occur because, after ingestion, they are transformed into carcinogens under the influence of the cytochrome p4501 enzyme system [86]. The carcinogenic mechanism is manifested in the fact that the carbon atoms of the alkyl NA molecule are hydroxylated by enzymes to produce hydroxyl NAs, which are denitrified by dealdehyde and denitrification process to form alkyl radicals. This phenomenon creates alkyl nucleic acids in the liver or cells, which are converted into alkylguanine that prevents DNA and RNA replication, and it is known that errors in DNA and RNA synthesis are caused by gene mutations, which can ultimately cause cancer in the human body [4].

A statistically significant positive trend was observed between increasing dietary intake of NDMA and the risk of stomach cancer. The association was stronger among women with low fruit and vegetable consumption. In the highest quintile of NDMA intake (>0.194 µg/day), the multivariable-adjusted hazard ratio (HR) for stomach cancer was 1.96 (95% CI: 1.08–3.58), indicating nearly a twofold increase in risk compared to the lowest quintile. These findings are consistent with results from four previous case–control studies, which reported a 1.4- to 7-fold increased risk of stomach cancer associated with a high intake of NDMA or other nitrosamines. Collectively, this evidence supports the potential role of nitrosamines in gastric carcinogenesis in humans [81]. Furthermore, a recent meta-analysis of English-language peer-reviewed studies published since 1990 examined the associations between nitrate, nitrite, and N-nitroso compounds and the risk of gastrointestinal cancers. The findings indicated that dietary nitrite intake was associated with an increased risk of gastric cancer, although the association was not significant across all sources of nitrate and NDMA. Notably, dietary intake of NDMA was significantly associated with an elevated risk of colorectal cancer (RR = 1.36, 95% CI: 1.18–1.58), particularly from processed meats such as bacon, hot dogs and sausages, which are naturally high in amine precursors [87].

Some of the most studied NAs are NDMA, NDEA, NPYR, NPIP, NDPA, and NDBA [65]. The International Agency for Research on Cancer (IARC) classifies NDMA, NDBA, NDEA, NDBA, NPIP and NPYR as potential carcinogenic compounds for humans (

Table 3. Overview of carcinogenic NAs according to the IARC classification [89].

Abbreviation	Name	CAS	Structure	IARC
NDMA	N-nitrosodimethylamine	62-75-9		2A
NDEA	N-nitrosodiethylamine	55-18-5		2A
NMU	N-Methyl-N-nitrosourea	684-93-5		2A
MNNG	N-Methyl-N′-nitro-N-nitrosoguanidine	70-25-7		2A
ENU	N-Ethyl-N-nitrosourea	759-73-9		2A
NPYR	N-nitrosopyrrolidine	930-55-2		2B
NPIP	N-nitrosopiperidine	100-75-4		2B
NDBA	N-nitrosodibutylamine	924-16-3		2B
NDPA	N-nitrosodipropylamine	621-64-7		2B
NMEA	N-Nitrosomethylethylamine	10595-95-6		2B
NDELA	N-Nitrosodiethanolamine	1116-54-7		2B
NSAR	N-Nitrososarcosine	13256-22-9		2B
NMVA	N-Nitrosomethylvinylamine	4549-40-0		2B
NMOR	N-Nitrosomorpholine	59-89-2		2B
MNPN	3-(N-Nitrosomethylamino)propionitrile	60153-49-3		2B
NMU	N-Methyl-N-nitrosourethane	615-53-2		2B

). The classification of NDBA, NPIP, and NPYR compounds falls under Group B2 [88], which indicates a probability of being carcinogenic, while NDMA and NDEA are classified as Group A2, which indicates a high possibility of carcinogenic compounds [23,65].

6. Legislation

Currently, there is no regulation within the European Union (EU) that determines the maximum permissible concentration of NAs in food; their presence is regulated only in elastomers, cosmetic products and toys. However, on 28 March 2023, the European Food Safety Authority (EFSA) published a scientific opinion entitled “Risk assessment of N-Nitrosamines in food” [14], where 10 quantified carcinogenic NAs were identified in processed meat (NDMA, NMEA, NDEA, NDPA, NDBA, NMA, NSAR, NMOR, NPIP and NPYR), which raises concerns for the health of consumers of all age groups. EFSA’s risk assessment highlighted uncertainties due to data limitations and concluded that exposure to these NAs poses a health concern. In response, the EU adopted Regulation (EU) 2023/2108 [90] to lower the level of NAs in cheese, meat and fish products, with the aim of reducing consumer exposure to carcinogenic NAs while maintaining protection from foodborne pathogens, given that the most common route of NA formation is from nitrite salts used as preservatives. However, there are gaps in knowledge and data on the presence of NAs in certain food categories, which highlights the need for further research and data collection.

Some countries have already introduced maximum levels (MLs) of concentration of NAs in their legislation such as the United States of America, where a limit of 10 ng/g of volatile NAs is prescribed for cured meat products. In China, limits of 4 µg/kg of N-nitrosodimetylamine (NDMA) for seafood and 3 µg/kg of NDMA for meat products are set because of their carcinogenicity and mutagenicity for humans at a very low concentration level [4]. In all of South America, with the exception of Chile, there is no permitted content of NAs, while in Chile, the MLs of NDMA in meat products are stipulated at 10 µg/kg. The Canadian Food Inspection Agency has set the MLs of NAs in cured meat products at 10 µg/kg for NDEA, NDPA, NIPI, NDMA and NDBA and 15 µg/kg for NPYR [5]. According to the World Health Organization (WHO), the daily limit of total NA is 10 µg/kg body weight [91].

Given the growing concern about exposure to NAs and their potential health risks, it is possible that the EU will consider setting MLs for NAs in food in the foreseeable future as ongoing scientific research continues to highlight the health risks associated with NAs in food, particularly in processed and cured meat products. According to the data on the content of NAs in individual meat products, there is a clear heterogeneity in their content not only in different but also in similar products. This heterogeneity may be the reason for the lack of regulation in the EU, despite the efforts of EFSA to collect information and data related to this subject [20]. Studies conducted by organizations such as EFSA are contributing to a better understanding of exposure levels to NAs and the associated health problems, which could prompt regulatory action. In addition, increased public awareness of food safety issues and consumer demand for safer food products, including the presence of NAs in food, may lead to increased pressure on regulatory authorities to act. The EU has a history of implementing regulations to address food safety concerns. For example, recent regulations have aimed to reduce exposure to contaminants such as tropane and ergot alkaloids in food. Given the potential health risks associated with NAs, it would not be unprecedented for the EU to set MLs for these compounds.

While there are indications that the EU may consider setting MLs for NAs in the future, the timing and specifics of any regulatory action will depend on various factors, including the availability of scientific evidence, monitoring activities and the prioritization of food safety issues within the EU regulatory agenda. Regular monitoring, risk assessments and public consultations are likely to play a role in shaping potential regulatory measures related to NAs in food.

7. Strategies to Reduce NA Formation

Given that NAs in processed meat products and food in general pose a high risk to human health, numerous studies have been conducted investigating the possibilities of reducing NA formation. For example, the presence of sodium chloride in meat products preserved with nitrites shows the potential to reduce the formation of NAs, although this effect is very low, according to some studies only at concentrations greater than 1.5% [24]. Sun et al. [92] investigated the inhibition of NAs in dried sausage using several species of Lactobacillus bacteria, and concluded that Lactobaillus curvatus had the most significant effect.

Some exogenous substances that effectively block NA synthesis include vitamins, phenols, sulfhydryls, quinones, spices, etc. [21]. Vitamins C and E play an important role in the inhibition of NAs. Vitamin E is a fat-soluble antioxidant and has the role of reducing nitrites to NO, while vitamin C has strong redox properties that protect cell membranes and participate in detoxification [93]. Polyphenolic compounds are frequently added to meat as inhibitors. Li et al. [94] concluded in their study that the addition of polyphenols and ascorbic acid to cured sausage effectively reduces nitrite residues and NAs. Wang et al. [95] added plant polyphenols and α-tocopherol to dry bacon and found an effect on biogenic amines, pH, and NDMA. Plant polyphenols such as those from green tea and grape seeds can also reduce nitrite residues [94]. The blocking mechanism of polyphenols is mainly attributed to the phenolic hydroxyl group, because it not only reacts with NO, but also reduces nitrous acid [96]. Liu et al. [97] used supercritical CO₂ extraction technology to extract flavonoids from corn silk. Research has shown that 500 µg/mL of flavonoid extract has a maximum nitrite effect of 88.1% at pH 3.0. Furthermore, soy phenols interfere with NO metabolism in an acidic environment, accelerating nitrite reduction [98].

In order to reduce human exposure to NAs from processed meat products, healthier preservation alternatives are being explored. More research has indicated the use of natural alternatives for nitrates and nitrites. Among the natural antimicrobial substances studied are lactoperoxidase, lactoferrin, lysozyme, ovotransferrin, avidin, nisin, bacteriocins, plants, spices and essential oils [17]. In their work, Deda et al. [99] discovered the effect of tomato paste on reducing nitrite levels in frankfurter sausage. Furthermore, spices have been proven to inhibit the formation of NAs, with pepper, star anise, cinnamon, cumin, etc., being the most prominent. For example, it has been proven that a high concentration of 15 g/kg black pepper can reduce the NPIP content in sucuk [100]. Also, the addition of onion and cumin spices during the production of fermented sausages can reduce the content of NDMA, NDEA, NMOR and NPIP, as well as the accumulation of biogenic amines [21]. Moreover, the addition of natural plant extracts showed good properties in removing nitrites, so the addition of carambola dietary fiber concentrates to the mixture of pork and turkey meat in the production of Vienna sausages increased the antioxidant capacity and significantly reduced the nitrite content [101]. Apart from being used as a food ingredient, garlic is also used as a medicinal plant, which, in addition to its therapeutic effects, also has anticancer, antiviral, antibacterial, and antifungal properties [102]. In the meat processing industry, garlic powder is used as an additive with antioxidant activity, and is also often used as a flavor enhancer [103]. Recent research has increasingly focused on the role of natural extracts in inhibiting the formation of N-nitrosamines (NAs) in food systems. Garlic (Allium sativum), for example, is rich in sulfur-containing compounds such as allicin (diallyl thiosulfinate), which has been shown to exert strong antioxidant and nitrite-reducing effects. These properties enable allicin and related organosulfur compounds to scavenge reactive oxygen species and interfere with nitrosation reactions, thereby limiting NA formation [104,105,106]. In addition to these functions, Chung et al. [107] found a reduction in NDMA formation in products with added garlic extract, while Choi et al. [104] found a significant effect of garlic juice under simulated gastric juice conditions on the NDMA blocking rate of 50.5%. Similarly, star anise (Illicium verum) contains a variety of phenolic compounds, including anethole, which possess both free radical-scavenging capacity and the ability to bind nitrite, reducing its reactivity in nitrosation pathways [108,109,110].

In addition to the listed additives in the production process, it is also possible to reduce NAs after the production process. Technologies such as irradiation and biodegradation can be used to degrade NAs, with gamma irradiation showing a particularly significant effect on their reduction. In their research, Mehrnia et al. [111] chose this radiation model as the best in the prevention of NAs, because there was a complete degradation and prevention of the NDEA reformation. UV irradiation of cured hams after two months resulted in a reduction in nitrite residues by about 26 to 70% after 11 months of storage [112]. Another study by Ahn et al. [113] investigated the effect of irradiation on pork sausages, where the sausages were vacuum-sealed immediately after stuffing, irradiated with 0, 5, 10 and 20 kGy and stored at 4 °C. It was concluded that the proportion of NDMA in vacuum-packed and NPYR in air-packed sausages was reduced at doses of 10 and 20 kGY. In another work, it was proven that the content of NDMA and NPYR in pepperoni and vegetable salami was also reduced after the irradiation process [114]. Biodegradable NAs are also very important and are currently the focus of research. For example, Rhodococcus RHA1 at concentrations of 20 to 2000 µg/L has good properties for degrading NAs such as NDMA, NDEA, NDPA, NPYR and NMOR [115].

8. Conclusions

The presence of NAs in meat products represents a persistent public health concern due to their well-documented carcinogenic potential. Despite significant progress in reducing NA levels through improved processing techniques and regulatory measures, dietary exposure remains relevant, especially in populations with high consumption of processed meats. This underscores the necessity of continued surveillance, harmonized analytical methods, and public awareness campaigns.

Looking ahead, the development of a comprehensive regulatory framework specific to NAs in food, similar to those already established for contaminants like mycotoxins and heavy metals, is essential. Such a framework should include clear maximum residue limits, routine monitoring, and mandatory reporting, particularly for compounds like NDMA and NDEA. A potential regulatory framework could be modeled after existing EU regulations on contaminants such as mycotoxins. It should include mandatory monitoring programs, harmonized sampling and testing protocols, transparent reporting systems, and periodic risk reassessment based on new toxicological and exposure data. Furthermore, collaboration with international bodies like WHO and IARC could foster a unified global approach towards setting maximum levels and mitigation strategies for NAs in meat products.

Integrating emerging technologies, such as more sensitive LC-MS/MS or GC-MS/MS platforms, will further enhance detection capabilities and risk assessment. Ultimately, coordinated international efforts will be crucial in minimizing consumer exposure and safeguarding public health.