Could Your Salted Fish Be Causing Cancer? The Hidden Nitrosamine Threat Every Consumer Should Know!

Analysis of Carcinogenic Nitrosamine Formation in Nitrite-Containing Salted Fish Products: Biochemical Mechanisms, Health Risks, Detection Methods, and Mitigation Strategies

 

ABSTRACT

 

Salted fish is a traditional food product with substantial economic and cultural significance in many Asian countries, including Indonesia. The salting and drying processes effectively extend the shelf life of fish by reducing water activity, thereby inhibiting the growth of spoilage microorganisms. However, in certain processing practices, sodium nitrite (NaNO₂) is still used as a food additive to preserve product color, inhibit the growth of anaerobic bacteria—particularly Clostridium botulinum—and improve product stability. The use of nitrite has raised significant food safety concerns because it can react with naturally occurring secondary and tertiary amines present in fish tissues to form N-nitroso compounds (NOCs), especially volatile nitrosamines such as N-nitrosodimethylamine (NDMA), which have been recognized as potent human carcinogens (IARC, 2012; WHO, 2023).

This review article comprehensively examines the chemical mechanisms underlying nitrosamine formation in salted fish, their metabolic bioactivation pathways in the human body, epidemiological evidence linking salted fish consumption with cancer, available laboratory analytical methods, current regulations governing nitrite use, and mitigation strategies applicable to the fisheries and food processing industries. The review was conducted through an extensive literature search of scientific publications retrieved from PubMed, Scopus, Web of Science, Google Scholar, Codex Alimentarius, the World Health Organization (WHO), the International Agency for Research on Cancer (IARC), the Food and Agriculture Organization (FAO), the European Food Safety Authority (EFSA), the Indonesian Food and Drug Authority (BPOM RI), as well as leading journals in food toxicology and analytical chemistry.

The literature indicates that nitrosamine formation is influenced by multiple factors, including nitrite concentration, the type of amine precursors, pH, heating temperature, salt concentration, water activity, and storage duration. The resulting nitrosamines undergo metabolic bioactivation primarily by cytochrome P450 enzymes, particularly CYP2E1, producing highly reactive alkylating intermediates capable of forming DNA adducts, inducing oxidative stress, triggering mutations in tumor suppressor genes such as TP53, and ultimately increasing the risk of several types of cancer, including nasopharyngeal carcinoma, gastric cancer, hepatocellular carcinoma, and esophageal cancer. Numerous epidemiological studies have consistently demonstrated a significant association between the consumption of traditional salted fish and an elevated incidence of nasopharyngeal carcinoma, particularly in East and Southeast Asian populations.

In conclusion, stringent control of nitrite use, implementation of alternative preservation technologies, strengthening food quality surveillance systems, and improving consumer education represent the principal strategies for reducing the risk of nitrosamine formation in salted fish products. Furthermore, additional research on nitrite-free preservation technologies and rapid nitrosamine detection methods remains essential to enhance food safety and protect public health.

 

Keywords: Nitrosamines; Sodium Nitrite; Salted Fish; N-Nitrosodimethylamine (NDMA); Nasopharyngeal Carcinoma; Carcinogens; Food Safety.

 

1. INTRODUCTION

 

Fish is one of the most valuable sources of animal protein due to its high nutritional value, excellent digestibility, and abundant content of omega-3 fatty acids, vitamins, and essential minerals. In many tropical countries, including Indonesia, fish represents a strategic food commodity that plays a crucial role in national food security. However, because fish is highly perishable, its quality deteriorates rapidly as a result of autolytic enzymatic activity, lipid oxidation, and microbial proliferation. Consequently, various preservation techniques have been developed to extend the shelf life of fish products (FAO, 2022).

Salting and drying are among the oldest fish preservation methods, having been practiced for thousands of years. These preservation techniques effectively reduce free water content and water activity (a_w), thereby significantly inhibiting the growth of spoilage bacteria, molds, and yeasts (Codex Alimentarius Commission, 2023). The resulting product, commonly known as salted fish, remains one of the most widely consumed traditional foods throughout Asia, including Indonesia, China, Vietnam, Thailand, the Philippines, Malaysia, and Japan.

In addition to extending shelf life, the salting process imparts distinctive sensory characteristics, including desirable flavor, aroma, and texture, which contribute substantially to consumer acceptance. Consequently, the salted fish industry has expanded from small-scale household production to large commercial enterprises. In Indonesia, thousands of micro- and small-scale enterprises depend economically on traditional salted fish production, making this commodity an important source of income and socioeconomic sustainability for coastal communities (Ministry of Marine Affairs and Fisheries of the Republic of Indonesia, 2023).

Nevertheless, increasing market demand for products with improved visual quality has encouraged some producers to incorporate food additives, including sodium nitrite (NaNO₂) and potassium nitrite (KNO₂), into the processing of salted fish. From a food technology perspective, nitrite provides several advantages, including stabilization of the characteristic reddish color of muscle tissue, inhibition of lipid oxidation, enhancement of flavor, and suppression of anaerobic pathogenic bacteria, particularly Clostridium botulinum, thereby improving product safety and shelf stability (Honikel, 2008; EFSA, 2023).

Despite these technological benefits, the use of nitrite also raises significant toxicological concerns. Under certain conditions, particularly in acidic environments or during high-temperature cooking, nitrite ions are converted into highly reactive nitrosating species. These reactive intermediates subsequently react with naturally occurring secondary and tertiary amines present in fish tissues to form N-nitroso compounds (NOCs). Among these compounds, volatile nitrosamines—including N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosopyrrolidine (NPYR), and N-nitrosopiperidine (NPIP)—have received considerable scientific attention because of their potent mutagenic and carcinogenic properties (IARC, 2010; WHO, 2023).

Nitrosamine formation in salted fish is not solely dependent on the presence of nitrite but is also strongly influenced by the naturally occurring trimethylamine oxide (TMAO) found in marine fish. During storage and fermentation, TMAO undergoes degradation into trimethylamine (TMA) and dimethylamine (DMA). These amines serve as the principal precursors for NDMA formation when they react with nitrosating agents derived from nitrite (Shahidi & Pegg, 1994). Consequently, the combination of abundant endogenous amines in marine fish and the addition of nitrite during processing creates favorable conditions for nitrosamine formation.

Over the past several decades, global concern regarding nitrosamines has increased substantially. The International Agency for Research on Cancer (IARC) has classified several nitrosamines as carcinogenic to humans or experimental animals. Furthermore, Chinese-style salted fish has been classified as a Group 1 carcinogen, indicating sufficient evidence of carcinogenicity in humans (IARC, 2012). This classification is supported by numerous epidemiological studies demonstrating a strong association between the consumption of traditional salted fish and an increased incidence of nasopharyngeal carcinoma (NPC), particularly in Guangdong Province (southern China), Hong Kong, Taiwan, Malaysia, Singapore, and several Southeast Asian countries.

Nasopharyngeal carcinoma is a squamous epithelial malignancy characterized by a unique geographical distribution. Although relatively uncommon in Europe and North America, its incidence is remarkably high in East and Southeast Asia. Besides genetic susceptibility and latent infection with Epstein–Barr virus (EBV), numerous epidemiological studies have consistently identified regular consumption of salted fish—especially during early childhood—as one of the most important environmental risk factors for NPC (Chang & Adami, 2006; IARC, 2012). Meta-analyses have shown that individuals who frequently consume salted fish exhibit a significantly higher risk of developing nasopharyngeal carcinoma than those with infrequent consumption, although the magnitude of risk varies according to consumption frequency, processing methods, and other lifestyle-related factors.

At the molecular level, nitrosamines are not intrinsically DNA-reactive compounds. Instead, they require metabolic bioactivation primarily by cytochrome P450 enzymes, particularly CYP2E1. This metabolic activation generates highly reactive electrophilic intermediates, including diazonium and methyldiazonium ions, which alkylate DNA and form mutagenic DNA adducts, especially O⁶-methylguanine. The accumulation of such DNA damage compromises genomic repair mechanisms, increases mutation rates in critical tumor suppressor genes such as TP53, activates cellular proliferation pathways, and ultimately promotes carcinogenesis (Hecht, 1999; Lijinsky, 1999).

In addition to carcinogenicity, excessive nitrite exposure has been associated with methemoglobinemia, endothelial dysfunction, increased oxidative stress, and several other toxicological effects. Consequently, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) has established an Acceptable Daily Intake (ADI) for nitrite of 0–0.06 mg/kg body weight per day, while individual countries have implemented maximum permissible nitrite residue limits according to the characteristics of specific food products (JECFA, 2017; EFSA, 2023).

Advances in analytical chemistry have enabled the detection of nitrite and nitrosamines at extremely low concentrations. In addition to the widely used Griess reagent colorimetric assay for routine nitrite determination, instrumental techniques such as High-Performance Liquid Chromatography (HPLC), Gas Chromatography–Mass Spectrometry (GC–MS), and Liquid Chromatography–Tandem Mass Spectrometry (LC–MS/MS) have become the gold-standard analytical methods for nitrosamine determination because of their superior sensitivity, selectivity, and low detection limits. These analytical capabilities provide essential scientific evidence for food safety surveillance and regulatory compliance at both national and international levels.

Given these considerations, the formation of nitrosamines in salted fish represents a multidisciplinary issue encompassing food biochemistry, toxicology, epidemiology, food processing technology, public health, and food safety regulation. Therefore, this review aims to provide a comprehensive analysis of the mechanisms underlying nitrosamine formation in nitrite-containing salted fish products, the factors influencing their formation, their metabolic bioactivation and carcinogenic mechanisms, the available epidemiological evidence, laboratory detection methods, current regulatory frameworks, and mitigation strategies that may reduce the associated public health risks.

 

2. METHODOLOGY

 

2.1 Study Design

This article was developed using a narrative literature review approach enriched with the principles of a Systematic Literature Review (SLR) based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA 2020) guidelines (Page et al., 2021). This approach was selected because it facilitates the integration of evidence from experimental studies, epidemiological investigations, toxicological research, biochemical studies, food technology literature, and international regulatory documents concerning the formation of nitrosamine compounds in nitrite-preserved salted fish products.

Unlike conventional systematic reviews, which generally address a single, narrowly defined research question, the present review aims to provide a comprehensive scientific synthesis of the relationship between nitrite use in salted fish products and the formation of N-nitroso compounds (NOCs), their metabolic bioactivation, toxicological effects, public health implications, laboratory analytical methods, food safety regulations, and mitigation strategies applicable to the fish processing industry.

This multidisciplinary approach was adopted because nitrosamine formation is a complex phenomenon involving food chemistry, microbiology, biochemistry, molecular toxicology, cancer epidemiology, food safety, analytical chemistry, and public health policy (Honikel, 2008; IARC, 2012).

2.2 Review Questions

This review was designed to address the following principal scientific questions:

1. What are the chemical mechanisms responsible for nitrosamine formation in nitrite-containing salted fish products?
2. Which factors influence nitrosamine formation during the processing and storage of salted fish?
3. How are nitrosamines metabolically bioactivated in the human body to induce genotoxic and carcinogenic effects?
4. How strong is the epidemiological evidence linking salted fish consumption with cancer, particularly nasopharyngeal carcinoma?
5. Which laboratory methods are most effective for detecting nitrite and nitrosamines in food products?
6. How do national and international regulatory frameworks govern the use of nitrite in fishery products?
7. Which mitigation strategies are most effective in minimizing nitrosamine formation in salted fish products?

2.3 Literature Search Strategy

Scientific literature was systematically retrieved through electronic searches of the following internationally recognized databases:

• PubMed/MEDLINE
• Scopus
• Web of Science
• ScienceDirect (Elsevier)
• SpringerLink
• Wiley Online Library
• Taylor & Francis Online
• Google Scholar

In addition to peer-reviewed scientific publications, official documents were obtained from the following organizations and regulatory agencies:

• World Health Organization (WHO)
• International Agency for Research on Cancer (IARC)
• Food and Agriculture Organization of the United Nations (FAO)
• Joint FAO/WHO Expert Committee on Food Additives (JECFA)
• European Food Safety Authority (EFSA)
• Codex Alimentarius Commission
• United States Food and Drug Administration (U.S. FDA)
• Indonesian Food and Drug Authority (BPOM RI)
• Ministry of Marine Affairs and Fisheries of the Republic of Indonesia

The inclusion of multiple scientific and regulatory sources was intended to ensure a comprehensive, up-to-date, and scientifically robust synthesis of current knowledge and regulatory developments.

2.4 Search Strategy

The literature search employed combinations of keywords using Boolean operators (AND, OR, and NOT) to maximize retrieval of relevant publications.

Whenever available, Medical Subject Headings (MeSH) terms were incorporated into the search strategy to improve search sensitivity and minimize inconsistencies arising from variations in scientific terminology.

2.5 Publication Period

The literature included in this review primarily comprised publications published between 2000 and 2025, with priority given to studies published within the past decade.

Nevertheless, several classical publications were retained because of their fundamental contributions to understanding nitrosation mechanisms and nitrosamine toxicology, including the pioneering works of:

• Magee and Barnes (1956)
• Lijinsky (1992, 1999)
• Hecht (1998, 1999)
• Honikel (2008)

These landmark studies continue to provide the scientific foundation for contemporary research on nitrosamines.

2.6 Inclusion Criteria

Studies were included in the review if they met the following criteria.

Publication type

• Original research articles
• Review articles
• Meta-analyses
• Systematic reviews
• International guidelines
• Government regulatory documents

Language

• English
• Indonesian

Research topics

Eligible studies addressed one or more of the following subjects:

• Nitrosamine formation
• Nitrite application in food products
• Salted fish
• Nitrosamine toxicology
• NDMA metabolism
• CYP2E1-mediated bioactivation
• Nasopharyngeal carcinoma
• Food safety
• Nitrite analytical methods
• Nitrite regulations

Scientific quality

Priority was given to publications appearing in peer-reviewed journals indexed in:

• Scopus
• Web of Science
• PubMed

2.7 Exclusion Criteria

Studies were excluded if they met any of the following conditions:

• Non-peer-reviewed publications.
• Full text unavailable.
• Duplicate publications.
• Studies focusing exclusively on nitrate without discussing nitrite or nitrosamines.
• Articles lacking a clearly described methodology.
• Reports providing low-quality scientific evidence.

2.8 Literature Selection According to PRISMA

The literature selection process followed the four principal stages recommended by the PRISMA 2020 guidelines (Page et al., 2021), including identification, screening, eligibility assessment, and final inclusion.

This structured selection process helped minimize selection bias and enhance the credibility and transparency of the review findings.

2.9 Data Extraction

Eligible studies were systematically evaluated using a standardized data extraction form containing the following variables:

Variable	Information Collected
Author	Author(s)
Publication year	Year of publication
Country	Study location
Study design	Experimental, epidemiological, or review
Fish species	Marine or freshwater fish
Product type	Salted fish, dried fish, fermented fish
Nitrite concentration	mg/kg
Nitrosamines	NDMA, NDEA, NPYR, NPIP
Analytical method	Griess assay, GC–MS, LC–MS/MS
Main findings	Concentration, risk assessment, and influencing factors

The extracted information was subsequently compared across studies to identify consistent patterns as well as variations attributable to differences in analytical methodology, sample characteristics, and food processing conditions.

2.10 Quality Assessment

The methodological quality of each publication was evaluated using the following criteria:

• Clarity of research objectives.
• Appropriateness of study design.
• Validity of laboratory analytical methods.
• Sample size.
• Statistical analysis.
• Potential sources of bias.
• Consistency with findings from previous studies.
• Overall level of scientific evidence.

Studies demonstrating higher methodological quality were assigned greater weight in the overall scientific synthesis.

2.11 Data Analysis and Synthesis

The extracted data were analyzed using a narrative synthesis approach because the substantial heterogeneity in study designs, laboratory methodologies, sample types, and measured outcomes precluded the performance of a quantitative meta-analysis.

The synthesis comprised six major components:

1. Chemical mechanisms, including the formation of nitrosonium ions, nitrosation of secondary amines, and the generation of volatile nitrosamines such as NDMA, NDEA, NPYR, and NPIP.
2. Factors influencing nitrosamine formation, including nitrite concentration, fish species, trimethylamine oxide (TMAO) content, pH, heating temperature, salt concentration, water activity, storage duration, and fermentation conditions.
3. Molecular toxicology, encompassing CYP2E1-mediated nitrosamine bioactivation, DNA adduct formation, oxidative stress, TP53 mutations, and carcinogenic mechanisms.
4. Epidemiological evidence, comparing findings from cohort studies, case-control studies, and meta-analyses investigating associations between salted fish consumption and nasopharyngeal carcinoma as well as gastrointestinal cancers.
5. Analytical technologies, including comparisons of the sensitivity, selectivity, limit of detection (LOD), limit of quantification (LOQ), and overall analytical performance of the Griess assay, HPLC, GC–MS, and LC–MS/MS.
6. Regulatory frameworks and mitigation strategies, comparing policies established by BPOM RI, the Codex Alimentarius Commission, EFSA, the U.S. FDA, and JECFA regarding nitrite regulation and strategies aimed at minimizing nitrosamine formation in food products.

Through this multidisciplinary approach, the present review is expected to provide a comprehensive understanding of nitrosamine formation in salted fish products, encompassing chemical mechanisms, molecular toxicology, epidemiological evidence, analytical methodologies, regulatory perspectives, and their broader implications for food safety and public health.

 

3. RESULTS AND DISCUSSION

 

3.1 Nitrite as a Food Additive in Salted Fish Products

Nitrite (NO₂⁻), primarily in the form of sodium nitrite (NaNO₂) and potassium nitrite (KNO₂), is one of the most widely used food additives in processed meat and fish products. Its principal technological functions include inhibiting the growth of anaerobic pathogenic bacteria, particularly Clostridium botulinum, preserving the characteristic reddish color of muscle tissue, retarding lipid oxidation, enhancing flavor, and extending product shelf life (Honikel, 2008; EFSA, 2023).

In traditional salted fish production, however, the use of nitrite is not considered part of the conventional preservation process. Traditional preservation relies primarily on high salt concentrations (typically exceeding 20%), which effectively reduce water activity (a_w) to levels below those required for the growth of most spoilage microorganisms (FAO, 2022). Nevertheless, increasing consumer demand for products with improved visual appearance has encouraged some manufacturers to incorporate nitrite into salted fish processing in order to maintain the desirable reddish coloration of fish muscle and enhance the perception of freshness.

Although nitrite provides important technological advantages, its application must be carefully controlled because it serves as a major precursor for the formation of N-nitroso compounds (NOCs). Numerous studies have demonstrated that NOC formation increases with increasing nitrite concentration, prolonged storage duration, and elevated processing temperatures (Cassens, 1997; Honikel, 2008). Consequently, the technological benefits of nitrite must be balanced against its potential health risks through appropriate manufacturing practices and regulatory oversight.

From a chemical perspective, nitrite is relatively stable under neutral conditions. However, under acidic conditions (pH < 3.5), such as those encountered in the human stomach or during certain stages of food processing, nitrite undergoes protonation to form nitrous acid (HNO₂). Nitrous acid subsequently decomposes into several highly reactive nitrosating species, including the nitrosonium ion (NO⁺), which is recognized as the principal nitrosating agent responsible for the formation of nitrosamines through reactions with secondary amines naturally present in fish tissues.

The propensity of nitrite to generate reactive nitrosating intermediates highlights the dual role of this food additive. While nitrite contributes substantially to microbial safety and product quality, inappropriate or excessive use may promote the formation of carcinogenic nitrosamines, particularly when suitable amine precursors, acidic conditions, and elevated temperatures coexist during food processing, storage, or digestion. Therefore, optimizing nitrite concentrations while maintaining microbiological safety remains a major challenge in modern food preservation and risk management.

3.2 Natural Nitrogenous Compounds in Fish as Nitrosamine Precursors

Marine fish naturally contain relatively high concentrations of non-protein nitrogenous compounds, many of which play essential physiological roles while simultaneously serving as potential precursors for nitrosamine formation during food processing and storage. Among these compounds, trimethylamine oxide (TMAO) is of particular importance because it functions as an osmolyte that maintains osmotic balance and stabilizes protein structure in marine fish inhabiting saline environments (Shahidi & Pegg, 1994).

Following harvest, TMAO undergoes enzymatic and microbial degradation during storage, fermentation, and processing. This degradation is catalyzed by endogenous enzymes as well as spoilage microorganisms, resulting in the formation of trimethylamine (TMA), dimethylamine (DMA), formaldehyde, and other volatile amines. These biochemical transformations not only contribute to the characteristic odor associated with deteriorating fish but also increase the availability of amine precursors capable of participating in nitrosation reactions.

The degradation pathway may be summarized as follows:

Trimethylamine oxide (TMAO) → Trimethylamine (TMA) + Dimethylamine (DMA) + Formaldehyde (HCHO)

Among these degradation products, dimethylamine (DMA) is considered the most important precursor for the formation of N-nitrosodimethylamine (NDMA), one of the most frequently detected and toxicologically significant volatile nitrosamines identified in salted and smoked fish products. Under acidic conditions or during thermal processing, DMA readily reacts with nitrosating agents derived from nitrite to generate NDMA, thereby substantially increasing the carcinogenic potential of the final food product.

In addition to DMA, fish tissues naturally contain several other secondary cyclic and aliphatic amines capable of undergoing nitrosation. These include diethylamine (DEA), pyrrolidine, piperidine, and morpholine, each of which can react with reactive nitrosating species to produce structurally distinct nitrosamines possessing different toxicological characteristics and carcinogenic potencies.

Table 1. Naturally Occurring Amines in Fish Products and Their Corresponding Nitrosamines

Amine Precursor	Nitrosamine Formed	Relative Carcinogenic Potential
Dimethylamine (DMA)	N-Nitrosodimethylamine (NDMA)	Very high
Diethylamine (DEA)	N-Nitrosodiethylamine (NDEA)	Very high
Pyrrolidine	N-Nitrosopyrrolidine (NPYR)	High
Piperidine	N-Nitrosopiperidine (NPIP)	High
Morpholine	N-Nitrosomorpholine (NMOR)	High

Among these compounds, NDMA and NDEA have received the greatest scientific attention because they exhibit exceptionally strong carcinogenic activity in experimental animal models and have been classified as probable or confirmed carcinogenic compounds based on extensive toxicological evidence. These nitrosamines have consistently demonstrated mutagenic activity in multiple biological systems and are capable of inducing tumors in several target organs following chronic exposure.

The abundance of endogenous amine precursors in marine fish explains why salted fish products possess a greater propensity for nitrosamine formation than many other processed foods. Importantly, nitrosamine formation depends not only on the presence of nitrite but also on the concentration and availability of precursor amines generated through protein degradation and TMAO metabolism. Consequently, marine fish species with naturally elevated TMAO levels generally exhibit a higher potential for NDMA formation than freshwater species.

Collectively, these findings indicate that the endogenous nitrogenous composition of fish constitutes a critical determinant of nitrosamine formation. Therefore, effective mitigation strategies should focus not only on controlling nitrite addition but also on minimizing the generation of precursor amines through appropriate handling practices, rapid chilling after harvest, optimized storage conditions, and processing techniques that limit enzymatic and microbial degradation of TMAO.

3.3 Chemical Mechanisms of Nitrosamine Formation

The formation of nitrosamines is a well-established chemical process involving the nitrosation of secondary amines by reactive nitrosating species generated from nitrite. This reaction represents the fundamental pathway responsible for the production of N-nitroso compounds (NOCs) in a wide variety of processed foods, particularly cured meat and salted fish products. Because marine fish naturally contain abundant secondary amines derived from trimethylamine oxide (TMAO) metabolism, the presence of nitrite creates favorable conditions for nitrosamine formation during processing, storage, cooking, and even digestion.

The nitrosation reaction is initiated when nitrite (NO₂⁻) is exposed to an acidic environment. Under low-pH conditions, nitrite is protonated to form nitrous acid (HNO₂) according to the following reaction:

NO₂⁻ + H⁺ ⇌ HNO₂

Nitrous acid is thermodynamically unstable and rapidly decomposes into several reactive nitrogen species, including dinitrogen trioxide (N₂O₃) and the highly electrophilic nitrosonium ion (NO⁺). Among these intermediates, the nitrosonium ion is recognized as the principal nitrosating agent responsible for transferring a nitroso group (–NO) to nucleophilic nitrogen atoms present in secondary amines.

The overall sequence of reactions may be summarized as follows:

NO₂⁻ + H⁺ → HNO₂ → NO⁺ (Nitrosonium ion)

Once formed, the nitrosonium ion readily reacts with secondary amines (R₂NH) to produce stable N-nitrosamines, as illustrated below:

R₂NH + NO⁺ → R₂N–N=O + H⁺

This reaction constitutes the central chemical mechanism underlying nitrosamine formation in food systems. Because secondary amines possess a nucleophilic nitrogen atom capable of accepting the electrophilic nitroso group, they are considerably more susceptible to nitrosation than primary amines, whereas tertiary amines generally undergo nitrosation only after prior metabolic or chemical transformation into secondary amines.

Among naturally occurring amines in marine fish, dimethylamine (DMA) is the most important precursor because it readily reacts with nitrosonium ions to produce N-nitrosodimethylamine (NDMA), one of the most potent carcinogenic nitrosamines identified in food products.

The reaction may be represented as follows:

(CH₃)₂NH + NO⁺ → (CH₃)₂N–N=O (NDMA)

Similarly, other naturally occurring amines—including diethylamine (DEA), pyrrolidine, piperidine, and morpholine—can undergo analogous nitrosation reactions, resulting in the formation of N-nitrosodiethylamine (NDEA), N-nitrosopyrrolidine (NPYR), N-nitrosopiperidine (NPIP), and N-nitrosomorpholine (NMOR), respectively. Although these compounds differ in chemical structure, they share a common N–N=O functional group, which is responsible for their characteristic biological and toxicological properties.

The efficiency of nitrosamine formation depends on several physicochemical factors. Acidic conditions markedly enhance nitrosonium ion generation, while elevated temperatures accelerate reaction kinetics by increasing both nitrite decomposition and amine reactivity. Consequently, high-temperature cooking methods such as frying, roasting, and grilling generally promote greater nitrosamine formation than boiling or steaming. In addition, prolonged storage increases the degradation of fish proteins and TMAO, thereby elevating the concentration of secondary amines available for nitrosation.

Conversely, several naturally occurring and added food constituents can inhibit nitrosamine formation. Reducing agents such as ascorbic acid (vitamin C) and erythorbate compete with secondary amines for reactive nitrosating species by converting nitrosonium ions and related intermediates into nitric oxide (NO), thereby preventing nitrosation. Likewise, numerous plant-derived polyphenols exhibit inhibitory effects through antioxidant activity and free radical scavenging mechanisms, reducing the availability of reactive nitrogen species involved in nitrosamine synthesis.

Overall, nitrosamine formation is governed by the dynamic interplay between nitrite availability, precursor amine concentration, environmental pH, processing temperature, storage conditions, and the presence of inhibitory compounds. Therefore, controlling these variables is essential for minimizing nitrosamine generation while preserving the technological benefits of nitrite in food preservation. A thorough understanding of these chemical mechanisms provides the scientific basis for developing effective mitigation strategies and improving the safety of nitrite-containing salted fish products.

3.4 Factors Affecting Nitrosamine Formation

Nitrosamine formation in salted fish products is a multifactorial process governed by the complex interactions among chemical composition, processing conditions, storage parameters, and the intrinsic characteristics of the raw material. Although the presence of nitrite and suitable amine precursors is a prerequisite for nitrosation, the extent of nitrosamine formation is strongly influenced by several physicochemical and biological factors. A comprehensive understanding of these factors is essential for designing effective mitigation strategies while maintaining the technological functions of nitrite in food preservation.

3.4.1 Effect of pH

Among the various factors influencing nitrosamine formation, pH is one of the most critical determinants. Nitrosation reactions occur most readily under acidic conditions because protonation of nitrite leads to the formation of nitrous acid (HNO₂), which subsequently generates highly reactive nitrosating species such as the nitrosonium ion (NO⁺).

Experimental studies have consistently demonstrated that the rate of nitrosamine formation increases dramatically within the pH range of 2–4, corresponding to the acidic environment of the human stomach. Consequently, nitrosation may occur not only during food processing but also after ingestion, when residual nitrite and naturally occurring amines are exposed to gastric acid. This phenomenon has important toxicological implications because endogenous nitrosamine formation may contribute to overall dietary exposure.

3.4.2 Effect of Temperature

Temperature is another major factor influencing nitrosamine formation. Elevated temperatures accelerate both nitrite decomposition and the kinetics of nitrosation reactions, thereby increasing the production of volatile nitrosamines.

Cooking methods involving intense heat, such as frying, roasting, and grilling, generally produce substantially higher concentrations of nitrosamines than moist-heat methods such as boiling or steaming. Several studies have reported that NDMA concentrations may increase several-fold after high-temperature frying, emphasizing the importance of thermal processing conditions in determining the final nitrosamine content of salted fish products.

The influence of temperature is particularly significant because thermal degradation of proteins and other nitrogen-containing compounds also increases the availability of secondary amines, thereby further promoting nitrosamine formation.

3.4.3 Effect of Storage Duration

Storage duration significantly influences nitrosamine formation through progressive biochemical and microbiological changes occurring within fish tissues. During prolonged storage, endogenous enzymes and spoilage microorganisms continue to degrade trimethylamine oxide (TMAO) and muscle proteins, leading to increased production of dimethylamine (DMA) and other volatile amines that serve as nitrosamine precursors.

Consequently, extended storage generally results in:

• increased degradation of TMAO;
• higher concentrations of DMA and other secondary amines;
• greater availability of nitrosatable substrates; and
• enhanced nitrosamine formation.

Proper refrigeration and reduced storage time are therefore essential for minimizing precursor accumulation and limiting subsequent nitrosation reactions.

3.4.4 Effect of Water Activity (a_w)

Water activity (a_w) plays a dual role in nitrosamine formation because it directly affects microbial growth, enzymatic activity, and chemical reaction rates.

Excessively high water activity favors microbial proliferation, resulting in increased protein degradation and enhanced formation of amine precursors. Conversely, extremely low water activity suppresses microbial metabolism and slows enzymatic degradation, thereby limiting precursor generation. Consequently, nitrosamine formation is influenced by the balance among moisture content, salt concentration, microbial activity, and storage conditions rather than by water activity alone.

Maintaining an optimal water activity is therefore an important consideration in controlling both microbial safety and chemical hazards in salted fish products.

3.4.5 Effect of Salt Concentration

Salt concentration is another important determinant of nitrosamine formation. High concentrations of sodium chloride effectively inhibit the growth of many spoilage microorganisms and pathogenic bacteria, thereby contributing to product preservation.

However, under certain fermentation or prolonged storage conditions, elevated salt concentrations may also accelerate protein denaturation and proteolytic degradation, resulting in increased production of free amino acids and secondary amines. Therefore, salt concentration exerts both inhibitory and promoting effects depending on the specific processing conditions and microbial ecology of the product.

Optimization of salt concentration is therefore necessary to achieve microbiological stability while minimizing the formation of nitrosamine precursors.

3.4.6 Effect of Fish Species

The intrinsic biochemical composition of fish species strongly influences their susceptibility to nitrosamine formation. Marine fish generally contain substantially higher concentrations of trimethylamine oxide (TMAO) than freshwater species because TMAO functions as an osmoprotectant in marine environments.

As a consequence, marine fish possess a greater capacity to generate dimethylamine (DMA) during storage and processing, making them inherently more susceptible to NDMA formation following exposure to nitrite. Differences in protein composition, lipid content, endogenous enzyme activity, and microbial communities among fish species may further contribute to variability in nitrosamine production.

3.4.7 Naturally Occurring Inhibitors of Nitrosamine Formation

In addition to factors promoting nitrosation, several naturally occurring compounds have been shown to inhibit nitrosamine formation. Ascorbic acid (vitamin C) is among the most effective inhibitors because it rapidly reduces reactive nitrosating species before they react with secondary amines. Likewise, vitamin E (α-tocopherol) suppresses lipid oxidation and indirectly decreases the formation of reactive intermediates associated with nitrosation.

Numerous plant-derived polyphenols, including those found in green tea, rosemary, turmeric, and other herbs and spices, also exhibit inhibitory effects through antioxidant activity, free radical scavenging, and interference with nitrosation pathways. These compounds have attracted considerable attention as natural alternatives for reducing nitrosamine formation in processed foods.

Table 2. Major Factors Influencing Nitrosamine Formation

Factor	Effect on Nitrosamine Formation
Nitrite concentration	Strongly increases
Low pH	Strongly increases
High processing temperature	Strongly increases
High-temperature frying	Strongly increases
Prolonged storage	Increases
High DMA concentration	Strongly increases
Vitamin C (ascorbic acid)	Inhibits
Vitamin E (α-tocopherol)	Inhibits
Polyphenols	Inhibits

Overall, nitrosamine formation is determined by the combined influence of precursor availability, environmental conditions, food composition, and processing practices. Since no single factor independently governs nitrosation, effective control requires an integrated approach encompassing optimized nitrite levels, appropriate thermal processing, controlled storage conditions, balanced salt concentrations, and the incorporation of natural nitrosation inhibitors. Such comprehensive risk management strategies are essential for minimizing nitrosamine formation while preserving product quality, microbiological safety, and consumer acceptance.

3.5 Bioactivation of Nitrosamines in the Human Body

Nitrosamines are relatively stable compounds upon ingestion and are not intrinsically reactive toward cellular macromolecules. Their carcinogenic potential arises only after metabolic bioactivation, a process that converts these chemically stable molecules into highly reactive electrophilic intermediates capable of interacting with DNA, proteins, and other critical cellular components. Consequently, nitrosamines are classified as procarcinogens, requiring enzymatic activation before exerting their genotoxic and carcinogenic effects (Hecht, 1999).

Following oral ingestion, nitrosamines are readily absorbed through the gastrointestinal tract and transported via the portal circulation to the liver, the principal site of xenobiotic metabolism. Hepatic biotransformation is mediated primarily by the cytochrome P450 (CYP450) enzyme system, with CYP2E1 recognized as the major isoenzyme responsible for the metabolic activation of low-molecular-weight nitrosamines, particularly N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA). Other CYP isoforms, including CYP2A6, CYP2B6, and CYP2D6, may also contribute to nitrosamine metabolism depending on the specific compound, tissue distribution, and species involved.

The bioactivation process begins with α-hydroxylation of the carbon atom adjacent to the N–nitroso group. This CYP2E1-catalyzed reaction generates an unstable α-hydroxynitrosamine intermediate, which rapidly undergoes spontaneous decomposition to produce highly reactive electrophilic species, including methyldiazonium ions, formaldehyde, and other alkylating intermediates.

The metabolic activation pathway of NDMA may be summarized as follows:

NDMA → CYP2E1-mediated α-hydroxylation → α-hydroxynitrosamine → Methyldiazonium ion + Formaldehyde + Reactive intermediates

Among these metabolites, the methyldiazonium ion is considered the principal ultimate carcinogenic species because of its exceptional electrophilic reactivity. This unstable intermediate readily transfers methyl groups to nucleophilic sites within biological macromolecules, particularly DNA bases, resulting in the formation of mutagenic DNA adducts that initiate the carcinogenic process.

Although the liver represents the primary site of nitrosamine bioactivation, CYP2E1 is also expressed in several extrahepatic tissues, including the esophagus, stomach, lungs, kidneys, and nasal epithelium. Consequently, local metabolic activation may occur in these organs, providing a mechanistic explanation for the tissue-specific carcinogenicity observed following chronic nitrosamine exposure. This organ-specific metabolism contributes to the elevated risks of cancers affecting the upper aerodigestive tract, liver, and gastrointestinal system.

The efficiency of nitrosamine bioactivation varies considerably among individuals due to genetic polymorphisms affecting CYP450 enzymes, differences in enzyme expression, age, nutritional status, alcohol consumption, smoking habits, concurrent medication use, and environmental exposures. Chronic alcohol consumption, for example, induces CYP2E1 expression, thereby enhancing nitrosamine activation and potentially increasing susceptibility to carcinogenesis. Likewise, tobacco smoke contains numerous nitrosamines and CYP2E1-inducing compounds, which may further amplify the biological effects of dietary nitrosamine exposure.

In addition to generating DNA-reactive alkylating agents, nitrosamine metabolism is accompanied by the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Excessive generation of these reactive molecules disrupts cellular redox homeostasis, induces oxidative stress, promotes lipid peroxidation, damages proteins and nucleic acids, and activates multiple inflammatory signaling pathways. Persistent oxidative stress further exacerbates genomic instability and enhances the likelihood of malignant transformation.

Under physiological conditions, the human body possesses several detoxification mechanisms capable of reducing nitrosamine toxicity. Phase II metabolic enzymes, including glutathione S-transferases (GSTs), facilitate the conjugation and elimination of certain reactive metabolites, while endogenous antioxidants such as glutathione, ascorbic acid (vitamin C), α-tocopherol (vitamin E), and antioxidant enzymes—including superoxide dismutase (SOD), catalase, and glutathione peroxidase—help neutralize oxidative damage generated during nitrosamine metabolism. However, prolonged or excessive exposure may overwhelm these protective mechanisms, resulting in cumulative cellular injury.

Overall, the carcinogenicity of nitrosamines depends not only on the amount ingested but also on the efficiency of their metabolic activation, the balance between activation and detoxification pathways, and the capacity of cellular defense systems to repair or eliminate DNA damage. Therefore, understanding the mechanisms of nitrosamine bioactivation provides an essential foundation for elucidating the molecular events leading to carcinogenesis and for developing preventive strategies aimed at reducing dietary exposure and improving food safety.

3.6 DNA Damage and Genetic Mutations

The carcinogenic potential of nitrosamines is primarily attributable to their ability to induce genotoxic DNA lesions following metabolic bioactivation. As discussed in the preceding section, CYP450-mediated metabolism of nitrosamines, particularly N-nitrosodimethylamine (NDMA), generates highly reactive alkylating intermediates, most notably the methyldiazonium ion. These electrophilic species readily attack nucleophilic sites within DNA, producing covalent modifications known as DNA adducts, which represent one of the earliest molecular events in chemical carcinogenesis (Hecht, 1999; Lijinsky, 1999).

Among the various DNA adducts produced by nitrosamine-derived alkylating agents, O⁶-methylguanine (O⁶-MeG) is considered the most biologically significant because of its strong mutagenic potential. This lesion forms when a methyl group is transferred to the oxygen atom at the O⁶ position of guanine, thereby altering its normal base-pairing properties. During DNA replication, O⁶-methylguanine preferentially mispairs with thymine instead of cytosine, resulting in G → A transition mutations, one of the most frequently observed mutation signatures associated with alkylating carcinogens.

The sequence of molecular events may be summarized as follows:

Nitrosamine bioactivation → Methyldiazonium ion formation → DNA alkylation → O⁶-methylguanine formation → Replication errors → Permanent gene mutations

Although cells possess an efficient repair mechanism through the enzyme O⁶-methylguanine-DNA methyltransferase (MGMT), which directly removes methyl groups from damaged guanine residues, excessive or prolonged nitrosamine exposure may overwhelm this repair capacity. Persistent accumulation of unrepaired DNA adducts increases genomic instability and substantially elevates the probability of mutation fixation during subsequent rounds of DNA replication.

In addition to O⁶-methylguanine, nitrosamine metabolism generates several other DNA lesions, including N7-methylguanine, O⁴-methylthymine, DNA strand breaks, abasic sites, and DNA–protein crosslinks. Although some of these lesions are less mutagenic individually, their cumulative effects contribute to chromosomal instability and impaired genomic integrity. Furthermore, oxidative metabolites generated during nitrosamine metabolism promote the formation of oxidative DNA lesions such as 8-hydroxy-2′-deoxyguanosine (8-OHdG), thereby amplifying DNA damage through mechanisms independent of direct alkylation.

The continuous generation of reactive oxygen species (ROS) during nitrosamine metabolism further aggravates genomic injury by inducing oxidative stress. Elevated ROS levels initiate lipid peroxidation, oxidize proteins, damage mitochondrial DNA, and activate multiple stress-responsive signaling pathways, including NF-κB, MAPK, and AP-1. Chronic oxidative stress also promotes persistent inflammation, creating a microenvironment that favors malignant transformation, tumor initiation, and cancer progression.

Accumulation of DNA damage ultimately affects genes responsible for maintaining genomic stability and regulating cell proliferation. Experimental and epidemiological studies have identified recurrent mutations in several cancer-associated genes following chronic nitrosamine exposure. Among these, the TP53 tumor suppressor gene is the most frequently affected. Loss or impairment of TP53 function compromises DNA damage surveillance, cell-cycle arrest, and apoptosis, thereby allowing genetically damaged cells to survive and proliferate.

Besides TP53, mutations have also been reported in several proto-oncogenes and signaling molecules, including:

• KRAS, leading to constitutive activation of proliferative signaling pathways;
• CTNNB1 (β-catenin), resulting in dysregulation of the Wnt/β-catenin signaling pathway;
• HRAS, which promotes uncontrolled cellular proliferation and survival.

Collectively, these genetic alterations disrupt the balance between oncogenic activation and tumor suppressor function, facilitating progressive accumulation of additional mutations and driving multistep carcinogenesis.

Beyond inducing direct genetic mutations, nitrosamine-derived DNA damage may also trigger epigenetic alterations, including aberrant DNA methylation, histone modifications, and dysregulated expression of non-coding RNAs such as microRNAs (miRNAs). These epigenetic changes can silence tumor suppressor genes or activate oncogenic pathways without altering the underlying DNA sequence, thereby further contributing to tumor development and progression.

Importantly, the biological consequences of nitrosamine-induced DNA damage depend on the dynamic interplay between DNA damage, repair efficiency, apoptosis, and immune surveillance. When DNA repair mechanisms successfully eliminate damaged cells, carcinogenesis can be prevented. However, if DNA lesions escape repair and affected cells evade apoptosis, permanent mutations become fixed within the genome and are propagated during cell division, initiating clonal expansion of genetically altered cells.

Overall, the available evidence indicates that nitrosamine-induced carcinogenesis is driven by a cascade of molecular events beginning with metabolic activation and DNA alkylation, followed by mutation accumulation, oxidative stress, genomic instability, and dysregulation of critical signaling pathways controlling cell growth and survival. These mechanisms provide a robust molecular basis for the strong epidemiological associations observed between chronic dietary nitrosamine exposure and increased risks of multiple human cancers, particularly nasopharyngeal, gastric, hepatic, esophageal, and colorectal malignancies.

3.7 Association Between Nitrosamines and Nasopharyngeal Carcinoma

The association between the consumption of nitrite-containing salted fish and nasopharyngeal carcinoma (NPC) represents one of the strongest and most extensively documented epidemiological relationships linking dietary exposure to nitrosamines with human cancer. Over the past several decades, numerous case–control studies, cohort investigations, and meta-analyses have consistently reported that habitual consumption of traditionally preserved salted fish is associated with a significantly increased risk of NPC, particularly in populations residing in East and Southeast Asia (Chang & Adami, 2006; IARC, 2012).

NPC is a malignant epithelial tumor arising from the mucosal lining of the nasopharynx. Unlike most head and neck cancers, NPC exhibits a striking geographical and ethnic distribution. The disease is relatively uncommon in Europe, North America, and most regions of Africa, but occurs at markedly higher incidence rates in Southern China—particularly Guangdong Province—as well as Hong Kong, Taiwan, Malaysia, Singapore, Indonesia, Vietnam, and several other Southeast Asian countries. This distinctive distribution suggests that NPC results from a complex interaction among genetic susceptibility, environmental exposures, dietary habits, and viral infection.

Among environmental risk factors, the consumption of Chinese-style salted fish has received particular attention. Based on extensive epidemiological evidence, the International Agency for Research on Cancer (IARC) has classified Chinese-style salted fish as a Group 1 carcinogen, indicating sufficient evidence of carcinogenicity in humans, primarily due to its association with nasopharyngeal carcinoma (IARC, 2012). This classification reflects the consistency of findings across diverse populations and study designs rather than the presence of a single carcinogenic compound.

Meta-analyses have demonstrated that individuals who regularly consume salted fish exhibit approximately 1.5- to 2.5-fold higher risks of developing NPC than those with infrequent or no consumption. The magnitude of risk varies according to consumption frequency, duration of exposure, methods of fish processing and preservation, and the age at which consumption begins. Notably, exposure during early childhood appears to confer the greatest increase in lifetime NPC risk, suggesting that the developing nasopharyngeal epithelium may be particularly susceptible to carcinogenic insults.

Several biological mechanisms have been proposed to explain the relationship between salted fish consumption and NPC. One of the most widely accepted hypotheses involves chronic exposure to volatile nitrosamines, including N-nitrosodimethylamine (NDMA) and related N-nitroso compounds generated during fish preservation, storage, cooking, and digestion. Following metabolic activation, these compounds produce DNA-reactive alkylating intermediates capable of inducing mutagenic DNA adducts within epithelial cells of the upper aerodigestive tract.

In addition to dietary exposure, volatile nitrosamines released during cooking may also be inhaled, resulting in direct contact with the nasopharyngeal mucosa. Repeated inhalational exposure has been proposed as an additional pathway contributing to local DNA damage and chronic epithelial injury, although the quantitative contribution of this route remains to be fully elucidated.

The development of NPC is now widely recognized as a multifactorial process in which nitrosamine exposure acts synergistically with other carcinogenic determinants. Among these, persistent latent infection with Epstein–Barr virus (EBV) is considered indispensable in the pathogenesis of the non-keratinizing subtype of NPC. Rather than acting independently, nitrosamine-induced genomic damage and EBV-mediated oncogenic mechanisms are believed to cooperate in promoting malignant transformation.

Nitrosamine-derived DNA damage may facilitate genomic instability, while EBV contributes through the expression of latent viral proteins and non-coding RNAs that interfere with apoptosis, immune surveillance, and cell-cycle regulation. Viral proteins such as latent membrane protein 1 (LMP1) activate multiple oncogenic signaling pathways—including NF-κB, PI3K/Akt, and JAK/STAT—thereby promoting cellular proliferation, angiogenesis, resistance to apoptosis, and chronic inflammation. The convergence of these pathways substantially accelerates the accumulation of molecular alterations required for tumor initiation and progression.

Host genetic susceptibility also plays an important role in determining individual risk. Variations in genes involved in carcinogen metabolism, DNA repair, immune regulation, and human leukocyte antigen (HLA) expression have been associated with differential susceptibility to NPC. Polymorphisms affecting enzymes such as CYP2E1, glutathione S-transferases (GSTs), and DNA repair proteins may influence both the metabolic activation of nitrosamines and the efficiency of repairing DNA damage, thereby modifying cancer risk among exposed individuals.

Although the association between salted fish consumption and NPC is supported by substantial epidemiological evidence, it is important to recognize that salted fish itself should not be regarded as the sole etiological factor. The carcinogenic risk is influenced by multiple variables, including nitrite concentration, levels of nitrosamine precursors, preservation techniques, cooking practices, dietary patterns, smoking, alcohol consumption, nutritional status, and genetic background. Consequently, individual susceptibility varies considerably across populations.

Beyond NPC, chronic exposure to dietary nitrosamines has also been associated with increased risks of several other malignancies, including gastric, esophageal, hepatocellular, colorectal, pancreatic, and bladder cancers, although the strength of evidence differs among cancer types. These observations reinforce the concept that nitrosamines are broad-spectrum chemical carcinogens capable of affecting multiple target organs following long-term exposure.

Overall, current epidemiological, toxicological, and molecular evidence strongly supports a causal relationship between chronic dietary exposure to nitrosamines derived from salted fish and an increased risk of nasopharyngeal carcinoma. This relationship exemplifies the complex interaction among environmental carcinogens, viral infection, host genetics, and dietary habits in human carcinogenesis. Accordingly, reducing dietary nitrosamine exposure through improved food processing technologies, strict control of nitrite use, enhanced food safety surveillance, and public health education should be regarded as important strategies for decreasing the global burden of NPC, particularly in high-incidence regions.

3.8 Analytical Methods for Nitrite and Nitrosamines

Reliable analytical methods are essential for assessing the safety of nitrite-containing foods and evaluating consumer exposure to carcinogenic nitrosamines. Modern food safety systems rely not only on the implementation of Good Manufacturing Practices (GMP) and Hazard Analysis and Critical Control Point (HACCP) programs but also on sensitive laboratory techniques capable of accurately quantifying both residual nitrite and trace concentrations of N-nitroso compounds (NOCs). Continuous advances in analytical instrumentation have substantially improved detection sensitivity, enabling the determination of nitrosamines at concentrations as low as the nanogram-per-kilogram (ng/kg) level (Pérez-Ortega et al., 2022; EFSA, 2023).

Analytical determination of nitrite and nitrosamines generally involves two distinct categories of methods. Nitrite is commonly quantified using colorimetric or spectrophotometric techniques because of its relatively high concentration in food matrices, whereas nitrosamines, which are typically present at trace levels, require highly selective chromatographic techniques coupled with mass spectrometric detection.

 

3.8.1 UV–Visible Spectrophotometry Using the Griess Method

The Griess assay remains one of the most widely employed methods for routine determination of nitrite in food products owing to its simplicity, rapidity, cost-effectiveness, and satisfactory analytical performance (Miranda et al., 2001). It is extensively applied in food control laboratories for monitoring compliance with regulatory limits on nitrite concentrations.

The analytical principle is based on the classical diazotization reaction. Under acidic conditions, nitrite reacts with sulfanilamide to form a diazonium salt. This intermediate subsequently undergoes an azo-coupling reaction with N-(1-naphthyl)ethylenediamine dihydrochloride (NED), producing a stable reddish-purple azo dye.

The overall reaction can be summarized as follows:

Nitrite + Sulfanilamide → Diazonium salt → + NED → Azo dye (λmax ≈ 540 nm)

The absorbance of the resulting azo compound is directly proportional to nitrite concentration and is quantified using a calibration curve generated from nitrite standards.

The Griess method offers several practical advantages, including:

• simple analytical procedure;
• rapid analysis;
• low operational cost;
• suitability for routine food quality monitoring; and
• adequate sensitivity for nitrite concentrations within the ppm range.

Despite these advantages, the method has an important limitation: it detects nitrite ions only and cannot identify or quantify nitrosamines that have already formed within the food matrix.

 

3.8.2 High-Performance Liquid Chromatography (HPLC)

High-performance liquid chromatography (HPLC) has become an important analytical tool for the determination of nitrite and particularly non-volatile nitrosamines. Separation is achieved through differential interactions between analytes, the stationary phase, and the mobile phase, allowing multiple compounds to be resolved within a single analytical run.

Depending on the analytical objective, HPLC systems may be coupled with several detectors, including:

• Ultraviolet (UV) detector;
• Diode array detector (DAD); and
• Fluorescence detector (FLD).

Compared with the Griess assay, HPLC provides superior selectivity, higher analytical accuracy, and the ability to separate multiple analytes simultaneously. Nevertheless, conventional HPLC generally exhibits lower sensitivity for volatile nitrosamines such as N-nitrosodimethylamine (NDMA) than chromatographic methods coupled with mass spectrometry.

 

3.8.3 Gas Chromatography–Mass Spectrometry (GC–MS)

Gas chromatography–mass spectrometry (GC–MS) is among the most widely accepted analytical techniques for determining volatile nitrosamines because of its exceptional sensitivity, selectivity, and molecular identification capability. It has become a reference method in food toxicology laboratories for monitoring nitrosamine contamination in processed foods.

The analytical procedure consists of two sequential stages. First, volatile compounds are separated according to their physicochemical properties using gas chromatography. Subsequently, individual compounds are ionized and identified by the mass spectrometer according to their characteristic mass-to-charge (m/z) ratios, providing highly specific molecular identification.

GC–MS is capable of simultaneously detecting several important volatile nitrosamines, including:

• N-nitrosodimethylamine (NDMA);
• N-nitrosodiethylamine (NDEA);
• N-nitrosopyrrolidine (NPYR);
• N-nitrosopiperidine (NPIP); and
• N-nitrosomorpholine (NMOR).

With detection limits reaching the low ng/kg range, GC–MS is widely regarded as one of the standard analytical techniques for food safety surveillance and toxicological investigations involving volatile nitrosamines.

 

3.8.4 Liquid Chromatography–Tandem Mass Spectrometry (LC–MS/MS)

Among currently available analytical techniques, liquid chromatography–tandem mass spectrometry (LC–MS/MS) is generally considered the gold standard for nitrosamine determination. The combination of high-performance liquid chromatography with tandem mass spectrometric detection provides outstanding analytical sensitivity, selectivity, and structural confirmation.

Major advantages of LC–MS/MS include:

• simultaneous determination of multiple nitrosamines within a single analytical run;
• ultra-low limits of detection, frequently below 1 ng/kg;
• excellent analytical specificity and accuracy;
• reduced matrix interference;
• minimal sample preparation for many food matrices; and
• the absence of derivatization requirements for numerous nitrosamines.

These characteristics make LC–MS/MS the preferred analytical platform for regulatory laboratories and research institutions involved in food safety surveillance throughout Europe, North America, Japan, Australia, and many other countries.

 

Table 3. Comparison of Analytical Methods for Nitrite and Nitrosamine Determination

Analytical Method	Primary Target	Typical Sensitivity	Major Advantages	Main Limitations
Griess spectrophotometry	Nitrite	ppm	Simple, rapid, inexpensive	Cannot detect nitrosamines
HPLC	Nitrite and selected nitrosamines	ppm–ppb	Good accuracy and compound separation	Lower sensitivity for volatile nitrosamines
GC–MS	Volatile nitrosamines	ppb–ng/kg	Excellent sensitivity and molecular specificity	More complex sample preparation
LC–MS/MS	Multiple nitrosamines	ng/kg	Highest sensitivity and selectivity; simultaneous multi-analyte analysis	High instrumentation and operating costs

The selection of an appropriate analytical technique depends on the objectives of the investigation, the nature of the food matrix, and the required detection limits. While the Griess assay remains suitable for routine monitoring of residual nitrite, chromatographic methods coupled with mass spectrometry are indispensable for comprehensive assessment of nitrosamine contamination. Among these, LC–MS/MS currently represents the most powerful analytical approach because of its exceptional sensitivity, selectivity, and capability to quantify multiple nitrosamines at trace concentrations. Continued advances in analytical instrumentation, sample preparation, and high-resolution mass spectrometry are expected to further improve the detection of emerging nitrosamines, thereby strengthening food safety surveillance and supporting more effective regulatory risk assessment.

3.9 Reported Nitrite and Nitrosamine Levels in Salted Fish Products

Numerous studies have demonstrated that the concentrations of residual nitrite and nitrosamines in salted fish products vary considerably according to fish species, preservation techniques, salt concentration, processing conditions, storage duration, and the analytical methods employed. This variability reflects the complex interplay between technological practices and the biochemical characteristics of the raw material, making it difficult to establish a universal concentration range applicable to all salted fish products.

In products manufactured using traditional salting methods without the intentional addition of synthetic nitrite, residual nitrite concentrations are generally low because preservation relies primarily on reduced water activity and high sodium chloride concentrations. Nevertheless, measurable amounts of nitrite may still be detected owing to endogenous nitrogen metabolism, microbial nitrate reduction, or environmental contamination during processing and storage.

Conversely, salted fish products prepared with added sodium nitrite (NaNO₂) may contain substantially higher residual nitrite concentrations, particularly when manufacturing practices are inadequately controlled or when excessive amounts of curing agents are used. Failure to comply with recommended nitrite levels not only increases residual nitrite concentrations but also enhances the likelihood of N-nitroso compound (NOC) formation during storage, cooking, and subsequent digestion.

Importantly, available evidence indicates that nitrosamine concentrations are not necessarily proportional to residual nitrite levels. Foods containing only moderate nitrite concentrations may nevertheless accumulate relatively high levels of nitrosamines when abundant precursor amines are present and favorable nitrosation conditions exist. Consequently, nitrosamine formation is determined by the interaction of multiple variables—including precursor availability, pH, thermal processing, storage duration, and oxidative conditions—rather than by nitrite concentration alone.

Marine fish species naturally contain higher concentrations of trimethylamine oxide (TMAO) than freshwater fish, providing a greater reservoir for the generation of dimethylamine (DMA) during storage and processing. As a result, marine salted fish generally exhibits a higher propensity for N-nitrosodimethylamine (NDMA) formation, particularly when exposed to acidic environments or high-temperature cooking. Similarly, smoked and fermented fish products may contain elevated concentrations of volatile nitrosamines because thermal processing and microbial activity promote both amine production and nitrosation reactions.

Several analytical surveys have consistently identified NDMA as the predominant nitrosamine detected in salted fish products, whereas N-nitrosodiethylamine (NDEA), N-nitrosopyrrolidine (NPYR), and N-nitrosopiperidine (NPIP) are generally detected at lower frequencies and concentrations. The relative abundance of individual nitrosamines depends largely on the composition of precursor amines within the fish tissue and on the specific processing conditions applied during preservation.

Table 4. Representative Nitrite and Nitrosamine Levels Reported in Processed Fish Products

Product	Nitrite (mg/kg)	Predominant Nitrosamine	Reference
Traditional salted fish	5–25	NDMA	Shahidi & Pegg (1994)
Smoked fish	10–40	NDMA, NPYR	Honikel (2008)
Fermented fish	8–30	NDMA	EFSA (2023)
Processed cured meat*	15–50	NDMA, NDEA	IARC (2012)

*Included for comparative purposes because cured meat products are among the most extensively studied food matrices regarding nitrite use and nitrosamine formation.

Note: The concentration ranges presented in this table represent a synthesis of values reported across multiple publications and are intended to provide an overall overview rather than data derived from a single study. Reported concentrations vary substantially depending on fish species, processing technology, formulation, storage conditions, geographical origin, and analytical methodology.

Interpretation of reported nitrosamine concentrations should therefore be undertaken with caution. Differences among studies frequently arise from variations in sample preparation procedures, extraction techniques, instrumental sensitivity, limits of detection (LOD), limits of quantification (LOQ), and quality assurance protocols. Moreover, advances in analytical instrumentation—particularly the introduction of gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS/MS)—have significantly improved the ability to detect trace concentrations of nitrosamines, resulting in greater analytical precision than earlier investigations.

From a public health perspective, chronic dietary exposure to low concentrations of nitrosamines may be of greater toxicological significance than occasional exposure to higher concentrations because carcinogenic risk is cumulative and depends on long-term intake. Consequently, continuous monitoring of both residual nitrite and nitrosamine concentrations is essential for ensuring food safety, evaluating compliance with national and international regulatory standards, and identifying opportunities for improving processing technologies.

Overall, the available evidence demonstrates that the occurrence of nitrosamines in salted fish products is highly variable and cannot be predicted solely from residual nitrite concentrations. Comprehensive risk assessment therefore requires simultaneous evaluation of nitrite content, precursor amine availability, processing conditions, storage practices, and analytical findings. This integrated approach provides a more accurate estimation of consumer exposure and supports the development of science-based strategies to minimize nitrosamine formation while maintaining the microbiological safety and quality of salted fish products.

3.10 Regulatory Framework for Nitrite Use

The use of nitrite as a food additive is subject to comprehensive regulatory oversight worldwide because its technological benefits must be carefully balanced against potential public health risks associated with the formation of carcinogenic N-nitroso compounds (NOCs). While nitrite remains indispensable for controlling microbial hazards—particularly Clostridium botulinum—its application is regulated under the principle that only the minimum amount necessary to achieve its intended technological function should be used.

International food safety authorities have established regulatory frameworks based on toxicological risk assessment, dietary exposure evaluation, and scientific evidence regarding the relationship between nitrite intake and adverse health outcomes. These regulations aim to minimize consumer exposure while preserving the microbiological safety and quality of processed foods.

3.10.1 Joint FAO/WHO Expert Committee on Food Additives (JECFA)

The Joint FAO/WHO Expert Committee on Food Additives (JECFA) has conducted extensive toxicological evaluations of nitrite and established an Acceptable Daily Intake (ADI) of 0–0.06 mg/kg body weight per day, expressed as nitrite ion (JECFA, 2017). This health-based guidance value was derived from long-term toxicological studies and incorporates uncertainty factors to ensure protection of the general population, including vulnerable groups.

The ADI is intended to represent the amount of nitrite that can be consumed daily over a lifetime without appreciable health risk. It serves as a fundamental reference for national regulatory agencies when establishing maximum permitted levels of nitrite in foods and evaluating dietary exposure from multiple food sources.

3.10.2 Codex Alimentarius Commission

The Codex Alimentarius Commission, jointly established by the Food and Agriculture Organization (FAO) and the World Health Organization (WHO), provides internationally recognized food standards intended to facilitate fair trade while protecting consumer health.

Rather than recommending universal maximum concentrations applicable to every food category, Codex emphasizes that nitrite should be used in accordance with the principle of Good Manufacturing Practice (GMP). Under this principle, nitrite should be added only at the lowest concentration necessary to achieve its technological purpose, including microbial inhibition, color stabilization, and product preservation.

The Codex approach recognizes that optimal nitrite concentrations may differ according to product formulation, processing technology, storage conditions, and microbiological risk. Consequently, manufacturers are encouraged to continuously optimize processing conditions to minimize residual nitrite and reduce the potential for nitrosamine formation.

3.10.3 European Food Safety Authority (EFSA)

The European Food Safety Authority (EFSA) has repeatedly reviewed the safety of nitrite as a food additive through comprehensive scientific risk assessments. Current EFSA evaluations conclude that, when used within established regulatory limits, the benefits of nitrite in preventing botulism generally outweigh the associated toxicological risks (EFSA, 2023).

Nevertheless, EFSA also emphasizes that every reasonable effort should be made to minimize the formation of nitrosamines during food production. Recommended measures include optimization of product formulations, strict control of processing temperatures, reduction of residual nitrite concentrations, incorporation of nitrosation inhibitors such as ascorbic acid or erythorbate, and continuous monitoring using validated analytical methods.

This risk-management strategy reflects the broader principle of As Low As Reasonably Achievable (ALARA), whereby exposure to potentially carcinogenic contaminants should be minimized whenever technically feasible without compromising food safety.

3.10.4 United States Food and Drug Administration (US FDA)

In the United States, the Food and Drug Administration (FDA) regulates the use of sodium and potassium nitrite under the Code of Federal Regulations (21 CFR). Maximum permitted concentrations vary according to food category and intended technological function. Compliance is enforced through manufacturing controls, routine inspection, laboratory surveillance, and adherence to current Good Manufacturing Practices (cGMP).

The FDA also encourages the incorporation of curing accelerators and nitrosation inhibitors, particularly ascorbate and erythorbate, which have been demonstrated to substantially reduce nitrosamine formation during processing of cured food products.

3.10.5 Indonesian Food and Drug Authority (BPOM RI)

In Indonesia, the use of nitrite as a food additive is regulated by the Indonesian Food and Drug Authority (BPOM RI) through BPOM Regulation No. 11 of 2019 on Food Additives, which specifies maximum permitted levels of nitrite according to food categories. Regulatory enforcement includes laboratory testing, post-market surveillance, and implementation of Good Processed Food Manufacturing Practices (Cara Produksi Pangan Olahan yang Baik; CPPOB).

The regulatory framework seeks to ensure that nitrite is used appropriately while minimizing potential health risks associated with excessive intake and nitrosamine formation. Continuous monitoring of food products in the marketplace remains an important component of national food safety programs.

3.10.6 International Harmonization and Future Perspectives

Despite broad international agreement regarding the technological necessity of nitrite, regulatory approaches continue to evolve in response to advances in toxicological research, analytical chemistry, and dietary exposure assessment. Improvements in high-sensitivity analytical techniques, particularly GC–MS and LC–MS/MS, have enabled more accurate measurement of trace nitrosamines, thereby strengthening scientific risk assessments and regulatory decision-making.

Increasing international harmonization among organizations such as JECFA, the Codex Alimentarius Commission, EFSA, the US FDA, and national regulatory authorities is expected to facilitate global trade while maintaining high standards of consumer protection. Future regulatory developments are also likely to encourage the adoption of alternative preservation technologies, natural antimicrobial compounds, and innovative processing methods capable of reducing dependence on nitrite without compromising microbiological safety.

Overall, effective regulation of nitrite use requires a balanced, science-based approach that integrates toxicological evidence, technological requirements, dietary exposure assessment, and continuous surveillance. Such an integrated regulatory framework is essential for minimizing nitrosamine formation, safeguarding public health, and ensuring the production of safe, high-quality salted fish products that comply with international food safety standards.

3.11 Mitigation Strategies for Reducing Nitrosamine Formation

Given the well-established carcinogenic potential of many N-nitroso compounds (NOCs), reducing nitrosamine formation has become a major objective in modern food preservation and safety management. Effective mitigation requires an integrated approach that combines technological innovation, food chemistry, microbiological control, quality assurance, and regulatory compliance. Because nitrosamine formation is influenced by numerous interacting factors—including nitrite concentration, precursor amines, pH, temperature, storage conditions, and processing methods—no single intervention can completely eliminate the risk. Instead, multiple complementary strategies should be implemented throughout the food production chain.

 

3.11.1 Reduction and Optimization of Nitrite Use

The most direct and effective strategy is to minimize the amount of sodium nitrite used while maintaining adequate antimicrobial protection and product quality. Nitrite should be applied according to the Good Manufacturing Practice (GMP) principle, whereby only the lowest concentration necessary to achieve its technological function is utilized.

Reducing residual nitrite significantly decreases the availability of nitrosating agents responsible for NOC formation. However, nitrite reduction must be carefully balanced against microbiological safety because inadequate curing may increase the risk of growth and toxin production by Clostridium botulinum and other pathogenic microorganisms.

Consequently, nitrite reduction should be accompanied by complementary preservation strategies, including improved sanitation, rapid chilling, optimized salt concentrations, appropriate packaging systems, and strict temperature control throughout processing and distribution.

 

3.11.2 Incorporation of Nitrosation Inhibitors and Natural Antioxidants

Among chemical mitigation strategies, the addition of ascorbic acid (vitamin C) and sodium erythorbate represents one of the most effective approaches for suppressing nitrosamine formation. These compounds inhibit nitrosation by reducing reactive nitrosating intermediates, including nitrosonium ions (NO⁺), before they can react with secondary amines (Mirvish, 1995).

In addition to conventional curing accelerators, increasing attention has been directed toward naturally occurring antioxidants with both free-radical scavenging activity and nitrosation-inhibitory properties. Numerous plant-derived bioactive compounds have demonstrated promising protective effects, including polyphenols, flavonoids, phenolic acids, and essential oil constituents.

Examples include extracts from:

• green tea (Camellia sinensis);
• rosemary (Rosmarinus officinalis);
• turmeric (Curcuma longa);
• garlic (Allium sativum);
• oregano (Origanum vulgare); and
• various culinary herbs and spices rich in antioxidant phytochemicals.

These natural compounds may reduce nitrosamine formation through multiple mechanisms, including scavenging reactive nitrogen species, inhibiting lipid oxidation, chelating transition metals, and stabilizing reactive intermediates. Their dual functionality as antioxidants and natural preservatives makes them attractive alternatives within clean-label food formulations.

 

3.11.3 Optimization of Processing Conditions

Processing parameters exert profound effects on nitrosamine formation and therefore represent important targets for technological intervention.

Several processing modifications have been shown to reduce nitrosamine generation, including:

• lowering cooking temperatures whenever technologically feasible;
• minimizing heating duration;
• replacing high-temperature frying or grilling with boiling or steaming;
• optimizing salt concentration to balance preservation efficacy and protein degradation;
• maintaining low storage temperatures to inhibit microbial activity and enzymatic degradation of trimethylamine oxide (TMAO) into dimethylamine (DMA); and
• reducing storage duration to limit precursor accumulation.

Control of pH is also important because nitrosation reactions are favored under acidic conditions. Optimizing formulation and processing variables can therefore substantially reduce the formation of reactive nitrosating species without compromising product safety.

 

3.11.4 Alternative Preservation Technologies

Recent advances in food technology have stimulated the development of preservation methods capable of reducing dependence on nitrite while maintaining microbiological stability and desirable sensory attributes.

Promising alternatives include:

• high-pressure processing (HPP);
• modified atmosphere packaging (MAP);
• vacuum packaging;
• cold plasma technology;
• ultraviolet (UV-C) treatment;
• ozonation;
• pulsed electric fields (PEF);
• natural antimicrobial coatings; and
• active packaging systems incorporating antioxidant or antimicrobial compounds.

These emerging technologies can reduce microbial contamination, delay spoilage, and extend shelf life while decreasing the need for chemical preservatives. Although many remain under industrial evaluation, they represent promising components of future nitrite-reduction strategies.

 

3.11.5 Natural Antimicrobial Agents

Considerable research has focused on replacing synthetic nitrite with naturally derived antimicrobial substances capable of inhibiting foodborne pathogens.

Potential alternatives include:

• chitosan and its derivatives;
• nisin and other bacteriocins;
• essential oils extracted from aromatic plants;
• fermented plant extracts;
• organic acids; and
• bioactive peptides produced by lactic acid bacteria.

These compounds possess antimicrobial activities against a broad spectrum of spoilage microorganisms and foodborne pathogens while generally exhibiting favorable toxicological profiles. Nevertheless, their effectiveness varies according to food composition, processing conditions, and storage environments, and further validation under commercial manufacturing conditions is still required.

 

3.11.6 Integrated Quality Assurance and Monitoring Systems

Mitigation of nitrosamine formation should be incorporated into comprehensive food safety management systems throughout the production chain.

Key preventive measures include:

• implementation of Good Manufacturing Practices (GMP);
• application of Hazard Analysis and Critical Control Point (HACCP) systems;
• continuous monitoring of residual nitrite concentrations;
• routine determination of nitrosamines using validated analytical methods;
• supplier quality assurance for raw materials;
• employee training in food safety practices; and
• documentation and traceability of production processes.

Routine surveillance using highly sensitive analytical techniques such as GC–MS and LC–MS/MS enables early detection of elevated nitrosamine concentrations and facilitates corrective actions before products reach consumers.

 

3.11.7 Future Perspectives

Future mitigation strategies are expected to integrate advances in food chemistry, biotechnology, nanotechnology, and digital food safety systems. The development of multifunctional natural preservatives, nanoencapsulated antioxidants, intelligent packaging capable of monitoring food quality in real time, biosensors for rapid nitrosamine detection, and artificial intelligence-assisted process optimization may substantially reduce nitrosamine formation while maintaining product safety and consumer acceptance.

Furthermore, systems biology approaches integrating metabolomics, predictive microbiology, and computational risk assessment are likely to improve understanding of nitrosamine formation pathways and facilitate the design of more effective intervention strategies. Such innovations will support the transition toward safer, more sustainable food preservation technologies with reduced reliance on synthetic curing agents.

Overall, successful mitigation of nitrosamine formation requires a multidisciplinary strategy encompassing optimized nitrite use, incorporation of natural inhibitors, improved processing technologies, advanced analytical surveillance, and rigorous quality management. The combination of these approaches offers the greatest potential for minimizing consumer exposure to carcinogenic nitrosamines while preserving the microbiological safety, nutritional quality, and commercial value of salted fish products.

3.12 One Health Approach for Nitrosamine Risk Control

The prevention of nitrosamine exposure extends beyond food manufacturing practices and should be addressed within the broader One Health framework, which recognizes the interdependence of human health, animal health, and environmental sustainability. Because nitrosamine formation is influenced by factors operating throughout the food production continuum—from aquaculture and raw material quality to food processing, distribution, and consumer handling—effective risk management requires coordinated actions across multiple sectors.

The One Health approach promotes interdisciplinary collaboration among food scientists, veterinarians, fisheries specialists, public health professionals, toxicologists, environmental scientists, regulatory authorities, and the food industry to ensure that food safety interventions are implemented comprehensively rather than in isolation. Such collaboration is increasingly important as global food systems become more complex and consumer demand for safe, minimally processed, and sustainable seafood products continues to increase.

3.12.1 Sustainable Aquaculture and Raw Material Quality

Nitrosamine risk management begins with the production of high-quality raw materials. The implementation of Good Aquaculture Practices (GAP) contributes to improved fish health, reduced microbial contamination, and enhanced product quality before processing. Proper feed management, biosecurity measures, disease prevention, water quality management, and responsible harvesting practices minimize spoilage and reduce the degradation of nitrogen-containing compounds that may later serve as nitrosamine precursors.

Rapid post-harvest handling, maintenance of the cold chain, and hygienic transportation are equally important because microbial decomposition of fish tissues promotes the conversion of trimethylamine oxide (TMAO) into dimethylamine (DMA) and other secondary amines involved in nitrosamine formation.

3.12.2 Food Processing and Industrial Quality Management

Within the processing sector, the application of Good Manufacturing Practices (GMP) and Hazard Analysis and Critical Control Point (HACCP) systems provides the foundation for controlling factors that influence nitrosamine formation. Critical control measures include optimization of nitrite concentrations, standardized salting procedures, temperature control during processing, hygienic manufacturing environments, and routine verification of processing parameters.

Food manufacturers should also adopt validated analytical monitoring programs for both residual nitrite and nitrosamines using sensitive techniques such as GC–MS and LC–MS/MS. Continuous quality assurance enables early identification of production deviations and facilitates corrective actions before products enter the market.

3.12.3 Public Health Surveillance and Regulatory Oversight

Government agencies play a central role in minimizing population exposure through regulatory enforcement, laboratory surveillance, and risk communication. National food safety authorities should establish coordinated monitoring programs capable of tracking nitrite residues, nitrosamine contamination, and compliance with regulatory standards across domestic and imported seafood products.

Risk assessment should incorporate not only contaminant concentrations but also dietary consumption patterns, cumulative exposure, vulnerable population groups, and regional differences in traditional food processing practices. Such surveillance provides the scientific basis for evidence-based policymaking and supports periodic revision of food safety regulations in response to emerging scientific knowledge.

3.12.4 Consumer Education and Risk Communication

Consumer awareness represents another essential component of nitrosamine risk reduction. Public education programs should emphasize appropriate food handling practices, healthy dietary patterns, and informed purchasing decisions. Educational initiatives may include recommendations to:

• consume salted fish products in moderation as part of a balanced diet;
• avoid excessive high-temperature frying or charring of cured products;
• maintain appropriate storage conditions after purchase;
• diversify dietary protein sources; and
• select products manufactured under recognized food safety standards.

Transparent risk communication based on scientific evidence can improve consumer confidence while preventing unnecessary public concern regarding the safe use of food preservatives.

3.12.5 Environmental Sustainability and Circular Food Systems

Environmental sustainability also contributes indirectly to nitrosamine prevention. Responsible fisheries management, reduction of post-harvest losses, efficient cold-chain infrastructure, and environmentally sustainable processing technologies improve raw material quality while decreasing spoilage-related biochemical changes that promote nitrosamine formation.

The transition toward circular food systems, resource-efficient processing, renewable energy utilization, and reduced food waste further supports sustainable seafood production while aligning food safety objectives with broader environmental and climate goals.

3.12.6 Research, Innovation, and International Collaboration

Continued scientific research remains essential for improving understanding of nitrosamine formation and identifying innovative mitigation strategies. Priority areas include the development of nitrite-free preservation technologies, natural antimicrobial compounds, advanced packaging systems, rapid biosensors for on-site nitrosamine detection, predictive models of nitrosation reactions, and artificial intelligence-assisted optimization of food processing parameters.

International collaboration among research institutions, regulatory agencies, and international organizations—including the Food and Agriculture Organization (FAO), the World Health Organization (WHO), the Codex Alimentarius Commission, the European Food Safety Authority (EFSA), and the Joint FAO/WHO Expert Committee on Food Additives (JECFA)—will facilitate harmonization of analytical methodologies, regulatory standards, and risk assessment frameworks. Such cooperation is particularly important as international trade in processed seafood products continues to expand.

3.12.7 Integrated One Health Framework

An effective One Health strategy for nitrosamine risk control should integrate preventive measures throughout the entire seafood production chain, including:

• implementation of Good Aquaculture Practices (GAP) to ensure high-quality raw materials;
• application of Good Manufacturing Practices (GMP) and HACCP during processing;
• optimization of nitrite use and incorporation of nitrosation inhibitors;
• routine laboratory surveillance using advanced analytical techniques;
• regulatory enforcement supported by science-based risk assessment;
• consumer education and transparent risk communication; and
• continuous research and international collaboration to promote innovation and regulatory harmonization.

By integrating these complementary components, the One Health framework provides a comprehensive strategy for reducing nitrosamine exposure while simultaneously protecting public health, ensuring animal welfare, preserving environmental sustainability, and strengthening the safety and global competitiveness of fishery products.

Ultimately, effective control of nitrosamine contamination requires coordinated action across the entire food system rather than isolated interventions at individual stages of production. A multidisciplinary One Health approach therefore offers the most sustainable pathway for minimizing carcinogenic risks associated with nitrite-containing salted fish products while supporting resilient food systems and long-term public health protection.

 

4. CONCLUSION

Nitrite remains an important food additive in certain processed fish products because of its technological functions, including inhibition of pathogenic microorganisms, preservation of desirable color, retardation of lipid oxidation, and extension of product shelf life. Nevertheless, the use of nitrite in salted fish products requires careful control because it can react with naturally occurring secondary and tertiary amines present in fish tissues, leading to the formation of N-nitroso compounds (NOCs). Among these, N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosopyrrolidine (NPYR), and N-nitrosopiperidine (NPIP) are of particular concern due to their well-documented mutagenic and carcinogenic properties. Several of these compounds have been classified by the International Agency for Research on Cancer (IARC) as carcinogenic or probably carcinogenic to humans based on substantial toxicological and epidemiological evidence.

This review demonstrates that nitrosamine formation is a multifactorial chemical process governed by the interaction of numerous variables, including residual nitrite concentration, precursor amine availability—particularly dimethylamine (DMA)—pH, thermal processing conditions, water activity, salt concentration, storage duration, and food processing methods. Marine fish generally possess a greater potential for nitrosamine formation than freshwater species because of their naturally higher concentrations of trimethylamine oxide (TMAO), which can be degraded into DMA during storage, microbial spoilage, or fermentation. Consequently, effective prevention of nitrosamine formation requires not only prudent control of nitrite use but also careful consideration of raw material characteristics and processing conditions throughout the production chain.

Following ingestion, nitrosamines undergo metabolic bioactivation primarily through the cytochrome P450 enzyme system, particularly CYP2E1, generating highly reactive electrophilic intermediates capable of forming DNA adducts such as O⁶-methylguanine. If these DNA lesions are not efficiently repaired, they may induce mutations in tumor suppressor genes, including TP53, disrupt cell-cycle regulation, promote oxidative stress, and ultimately initiate carcinogenesis. These molecular mechanisms provide strong biological plausibility for the observed association between chronic nitrosamine exposure and increased cancer risk.

From an epidemiological perspective, evidence derived from case-control studies, cohort studies, and meta-analyses consistently supports an association between habitual consumption of traditional salted fish and an increased risk of nasopharyngeal carcinoma (NPC), particularly among populations in East and Southeast Asia. The risk appears to be greater when exposure begins during childhood or continues over prolonged periods. In addition to NPC, dietary nitrosamine exposure has also been associated with elevated risks of gastric, hepatic, esophageal, colorectal, and pancreatic cancers, although the magnitude of these associations is influenced by exposure dose, host genetic susceptibility, Epstein–Barr virus (EBV) infection status, smoking habits, alcohol consumption, and other environmental and lifestyle factors.

Advances in analytical chemistry have markedly enhanced the detection and quantification of nitrite and nitrosamines in food products. The Griess spectrophotometric assay remains an appropriate method for routine determination of residual nitrite because of its simplicity, rapidity, and low analytical cost. In contrast, gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) represent the most sensitive and selective techniques for identifying and quantifying multiple nitrosamines at trace concentrations. Continued development of faster, more sensitive, and cost-effective analytical technologies remains essential for strengthening food safety surveillance and regulatory compliance.

International organizations, including the Joint FAO/WHO Expert Committee on Food Additives (JECFA), the Codex Alimentarius Commission, the European Food Safety Authority (EFSA), and the Indonesian Food and Drug Authority (BPOM RI), have established science-based regulations governing the use of nitrite as a food additive. However, the effectiveness of these regulatory frameworks ultimately depends on industry compliance, robust food safety monitoring systems, and the availability of accredited laboratories capable of accurately determining residual nitrite and nitrosamine concentrations.

Practical Implications

Based on the evidence synthesized in this review, several practical strategies may substantially reduce the risk of nitrosamine formation in salted fish products:

1. Optimize nitrite use in accordance with the principles of Good Manufacturing Practice (GMP) by applying only the minimum concentration required to achieve effective preservation.
2. Promote alternative preservation technologies, including plant-derived extracts, bacteriocins, chitosan, and non-thermal preservation methods, to reduce reliance on synthetic nitrite.
3. Incorporate nitrosation inhibitors, particularly ascorbic acid (vitamin C), sodium erythorbate, and natural antioxidants that have demonstrated efficacy in suppressing nitrosamine formation.
4. Optimize thermal processing conditions by avoiding excessive heating and high-temperature frying, both of which accelerate nitrosation reactions.
5. Strengthen education and training for food producers and consumers regarding the health risks associated with excessive nitrite use and the importance of proper hygiene and sanitation throughout food processing.
6. Enhance food safety surveillance through routine monitoring of residual nitrite and nitrosamine concentrations by governmental and accredited analytical laboratories.
7. Adopt a One Health approach that integrates food safety, public health, animal health, and environmental sustainability across the entire seafood production chain.

Study Limitations

This article is based on a narrative review of published scientific literature; therefore, its conclusions rely on the synthesis of previously reported evidence rather than newly generated experimental data. Considerable heterogeneity exists among available studies with respect to study design, fish species, processing technologies, analytical methodologies, product characteristics, and regulatory frameworks across different countries. Consequently, interpretation of the available evidence should be undertaken with appropriate caution.

Furthermore, much of the current scientific literature concerning nitrosamines has focused primarily on processed meat products, whereas studies specifically investigating traditional salted fish—particularly those produced in Indonesia and other Southeast Asian countries—remain comparatively limited. Additional high-quality investigations are therefore needed to improve the evidence base for seafood-specific risk assessment.

Future Research Directions

Future research should focus on several priority areas to improve understanding and control of nitrosamine formation in salted fish products:

• development of nitrite-free preservation technologies capable of effectively controlling pathogenic microorganisms while maintaining product quality;
• exploration of natural antioxidants and bioactive compounds as effective inhibitors of nitrosamine formation;
• identification and validation of biomarkers of nitrosamine exposure in populations with high consumption of salted fish;
• application of quantitative microbial and chemical risk assessment models to estimate population exposure and health risks;
• integration of omics technologies, including genomics, proteomics, metabolomics, and transcriptomics, to elucidate the molecular mechanisms underlying nitrosamine-induced carcinogenesis; and
• development of rapid detection systems, including biosensor- and nanotechnology-based analytical platforms, for real-time monitoring of nitrite and nitrosamine contamination in food products.

As research in food toxicology, analytical chemistry, molecular epidemiology, and food processing technologies continues to advance, more effective strategies for minimizing nitrosamine formation are expected to emerge. These innovations will contribute to safer preservation practices while maintaining the nutritional quality, sensory characteristics, and commercial competitiveness of salted fish products in both domestic and international markets. Ultimately, integrating scientific innovation with evidence-based regulation and the One Health framework will be essential for protecting public health and ensuring the long-term sustainability of the global seafood industry.

 

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