Nitrosamines crisis in pharmaceuticals-Insights on toxicological implications,root causes and risk assessment:A systematic review

The presence of N-nitroso compounds,particularly N-nitrosamines,in pharmaceutical products has raised global safety concerns due to their significant genotoxic and mutagenic effects.This systematic review investigates their toxicity in active pharmaceutical ingredients(APIs),drug products,and pharmaceutical excipients,along with novel analytical strategies for detection,root cause analysis,reformulation strategies,and regulatory guidelines for nitrosamines.This review emphasizes the molecular toxicity of N-nitroso compounds,focusing on genotoxic,mutagenic,carcinogenic,and other physiological effects.Additionally,it addresses the ongoing nitrosamine crisis,the development of nitrosamine-free products,and the importance of sensitive detection methods and precise risk evaluation.This comprehensive overview will aid molecular biologists,analytical scientists,formulation scientists in research and development sector,and researchers involved in management of nitrosamine-induced toxicity and promoting safer pharmaceutical products.

Hepatoprotective effect of Holothuria leucospilota methanolic extract on dimethyl nitrosamine-induced hepatotoxicity in rats

Background:Complementary medicine is an interesting field for extracting bio-active compounds from various plant and animal sources.The hepatoprotective effect of the methanolic extract of a species of sea cucumber called Holothuria leu-cospilota in an animal model of liver cancer caused by dimethyl nitrosamine(DMN)was studied.Methods:Wistar female rats were randomly divided into five groups(n=12):control(intact),positive control(received 1%DMN[10 mg/kg/week,intraperitoneally]for 12 weeks),and three treatment groups(received 50,100,and 200 mg/kg/day H.leu-cospilota extract orally for 12 weeks along with intraperitoneal administration of 1%DMN[10 mg/kg/week]).In all groups,ultrasound was performed on the liver every week to check its density.Blood sampling and liver isolation were performed on three occasions,at 4,8,and 12 weeks,to check liver enzymes and the histopathological condition of the liver tissue(every week,four animals from each group were randomly selected).Results:Liver density changes were evident from the eighth week onward in the positive control group.Histopathological results indicated pathologic changes in the positive control group after 4 weeks.The increase in liver enzymes in the posi-tive control group was significantly different from that in the treatment and control groups.Conclusions:We demonstrated the hepatoprotective effect of H.leucospilota on DMN-induced liver damage in rats using biochemical and histological parameters and ultrasonography.More additional research(in silico or in vitro)is needed to find the exact mechanism and the main biological compound in H.leucospilota.

Intake of Volatile N-nitrosamines and Their Ability to Exogenously Synthesize in the Diet of Inhabitants from High-risk Area of Esophageal Cancer in Southern China

Objective Nan'ao County in Guandong Province is a high-risk area of esophageal cancer in Southern China. Of the suspected etiological factors in the environment, N-nitrosamines and their precursors have received the greatest attention. Methods Sixty samples of the diet ingested by the inhabitants were collected and detected for volatile N-nitrosamines and their precursors. Five N-nitrosamines detected by Gas Chromatography-Thermal Energy Analyzer were N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosopyrrolidine, N-nitrosopiperidine and N-nitrosomethyl-benzylamine. Results The average content of 5 volatile N-nitrosamines in the diet was 312.0 μg/kg (median). The daily intake of the nitrosamines was 286.5 μg/head/day. Only the ability to exogenously synthesize N-nitrosopiperidine was powerful among 5 volatile N-nitrosamines. By a computerized stepwise regression analysis and curve fitting, we studied the correlation among the nitrosamines, the precursors and the major food items in the samples. Conclusion It demonstrated that a relatively high content of volatile N-nitrosamines was present in the diet collected in the area.

Nitrate-nitrite-nitrosamines exposure and the risk of type 1 diabetes: A review of current data

The potential toxic effects of nitrate-nitrite-nitrosamine on pancreatic β cell have remained a controversial issue over the past two decades. In this study, we reviewed epidemiological studies investigated the associations between nitrate-nitrite-nitrosamines exposure, from both diet and drinking water to ascertain whether these compounds may contribute to development of type 1 diabetes. To identify relevant studies, a systematic search strategy of Pub Med, Scopus, and Science Direct was conducted using queries including the key words "nitrate", "nitrite", "nitrosamine" with "type 1 diabetes" or "insulin dependent diabetes mellitus". All searches were limited to studies published in English. Ecologic surveys, case-control and cohort studies have indicated conflicting results in relation to nitrate-nitrite exposure from drinking water and the risk of type 1 diabetes. A null, sometimes even negative association has been mainly reported in regions with a mean nitrate levels < 25 mg/L in drinking water, while increased risk of type 1 diabetes was observed in those with a maximum nitrate levels > 40-80 mg/L. Limited data are available regarding the potential diabetogenic effect of nitrite from drinking water, although there is evidence indicating dietary nitrite could be a risk factor for development of type 1 diabetes, an effect however that seems to be significant in a higher range of acceptable limit for nitrate/nitrite. Current data regarding dietary exposure of nitrosamine and development of type 1 diabetes is also inconsistent. Considering to an increasing trend of type 1 diabetes mellitus(T1DM) along with an elevated nitrate-nitrite exposure, additional research is critical to clarify potential harmful effects of nitrate-nitritenitrosamine exposure on β-cell autoimmunity and the risk of T1DM.

Analysis of four tobacco-specific nitrosamines in mainstream cigarette smoke of Virginia cigarettes by LC-MS/MS

An improved method was developed for the determination of the four major tobacco-specific nitrosamines（TSNAs） in mainstream cigarette smoke. The new method offers decreased sample preparation and analysis time as compared to traditional methods. This method uses isotope dilution liquid chromatography coupled with a tandem mass spectrometer with electrospray ionization and is significantly more sensitive than traditional methods. It also shows no evidence of artifactual formation of TSNAs. Sample concentrations were determined for four TSNAs in mainstream smoke using two isotopically labeled TSNAs analogues as internal standards. Mainstream smoke was collected on an industry standard 44-ram Cambridge filter pad, extracted with 0.1 mol/L ammonium acetate, purified by solid-phase extraction, and analyzed without further sample cleanup. The analytical column is a 3.9 mm×150 mm Waters Symmertry Shield RP18 column and volume fraction of the mobile phase is 50% methanol, 50% water containing 0.1% acetic acid. The results show that the linear range is 0.5-100.0 mg/L except for N-nitrosoanabasine （NAB） from 0.25 to 50.0 mg/L. The limits of detection are 0.1 mg/L for N-nitrosonornicotine （NNN）, 0.08 mg/L for 4-（methylnitrosamino）-1- （3-py-ridyl）-1-butanone （NNK）, 0.05 mg/L for N-nitrosoanatabine （NAT） and 0.06 mg/L for NAB. The recoveries of the four TSNAs are from 90.2% to 105.7%.

Ultra-Sensitive LC-MS/MS Method for the Trace Level Quantification of Six Potential Genotoxic Nitrosamine Impurities in Telmisartan

Nitrosamine impurities are potentially genotoxic which are considered under cohort of concern as per ICH M7 guidelines and need to be controlled at trace levels during quantification in drug substances and drug products for safe human consumption. Recent regulatory requirements also suggest the need to have highly sensitive analytical methods for the accurate quantification of Nitrosamine impurities. In this paper we have presented simple, rapid and ultra-sensitive LC-MS/MS method for six potential genotoxic nitrosamine impurities: N-Nitroso dimethyl amine (NDMA), N-Nitroso diethyl amine (NDEA), N-Nitroso Ethyl Iso propylamine (NEIPA), N-Nitroso-N-methyl-4-aminobutyric acid (NMBA) N-Nitroso diisopropylamino (NDIPA) and N-Nitroso dibutyl amine (NDBA) with a LOQ of 0.004 ppm. Chromatographic separation is achieved using Zorbax SB C18 150 × 3.0 mm, 3.5 μ column with 0.1% formic acid in water as mobile phase A and 0.1% formic acid in methanol as mobile phase B at a flow rate of 0.3 ml/min using gradient mode of elution at a total run time of 18 minutes. Six nitrosamine impurities are successfully ionized and quantified in positive mode of atmospheric pressure chemical ionization (APCI) using multiple reaction monitoring (MRM). Method validation is performed as per ICH guidelines evaluating the limit of quantification and detection and found to give good S/N ratios with good linearity range of 0.002 - 2 ppm with regression coefficient >0.99 for all the six nitrosamine impurities. Method recoveries are established using three-step sample preparation protocol and are found to be satisfactory within 80% - 120%. The method can be used routinely applied for the detection of Nitrosamines in Telmisartan at a concentration of 1.5 ng/ml (0.03 ppm with respect to telmisartan concentration of 50 mg/ml).

A New Automated Method and Sample Data Flow for Analysis of Volatile Nitrosamines in Human Urine

Volatile nitrosamines (VNAs) are a group of compounds classified as probable (group 2A) and possible (group 2B) carcinogens in humans. Along with certain foods and contaminated drinking water, VNAs are detected at high levels in tobacco products and in both mainstream and side-stream smoke. Our laboratory monitors six urinary VNAs—N-nitrosodimethylamine (NDMA), N-nitrosomethylethylamine (NMEA), N-nitrosodiethylamine (NDEA), N-nitrosopiperidine (NPIP), N-nitrosopyrrolidine (NPYR), and N-nitrosomorpholine (NMOR)—using isotope dilution GC-MS/ MS (QQQ) for large population studies such as the National Health and Nutrition Examination Survey (NHANES). In this paper, we report for the first time a new automated sample preparation method to more efficiently quantitate these VNAs. Automation is done using Hamilton STARTM and Caliper StaccatoTM workstations. This new automated method reduces sample preparation time from 4 hours to 2.5 hours while maintaining precision (inter-run CV < 10%) and accuracy (85% - 111%). More importantly this method increases sample throughput while maintaining a low limit of detection (<10 pg/mL) for all analytes. A streamlined sample data flow was created in parallel to the automated method, in which samples can be tracked from receiving to final LIMs output with minimal human intervention, further minimizing human error in the sample preparation process. This new automated method and the sample data flow are currently applied in bio-monitoring of VNAs in the US non-institutionalized population NHANES 2013-2014 cycle.

SYNTHESES OF N- METHYL-^(14)C BENZYL AMINE AND N- METHYL-^(14)C BENZYL NITROSAMINE

The syntheses of N-Methyl-14C benzyl amine and N-Methyl-14C benzyl nitrosamine are reported. Specific activities were approximately 920 MBq/mmol. Chemical and radiochemical purity checked by HPLC were more than 95%.

Association of a Tobacco-specific Nitrosamine Carcinogen with Urinary Cotinine,Urinary Sodium Excretion,and Total Energy Intake in Adolescents and Children

This study investigated the association of a tobacco-specific nitrosamine carcinogen,4-(methylnitrosamino)-1-(3-pyridy1)-I-butanol(NNAL)with urinary cotinine(uCot),urinary sodium(uNa)excretion,systolic blood pressure(sBP),and total energy intake in adolescents and children in relation to the subjects'age.A total of 790 subjects aged 6-19 years were evaluated.NNAL,uCot,corrected NNAL(cNNAL),the NNAL/uCot ratio,uNa,sBP,and nutrient intake were measured.A strong association between uCot and cNNAL was observed in children who were 11 years of age(r=0.881,P<0.001);however,no significant association was noted in adolescents who were 19 years of age.The uNa level was significantly higher(133.9 mmol/L vs.107.8 mmol/L,P<0.001)and sBP was significantly lower(105.3 mmHg vs.110.6 mmHg,P=0.012)in adolescents with elevated NNAL than in those without elevated NNAL.NNAL was significantly higher in subjects with increased uNa excretion than in those without increased uNa excretion.NNAL was positively correlated with uNa(r=0.183,P<0.001)and negatively correlated with sBP(r=0.142,P<0.001).Non-smokers with elevated NNAL/uCot ratios had significantly lower total energy intake than those without elevated NNAL/uCot ratios(1729.0 kcal/day vs.1911.0 kcal/day,P=0.008).The relationship between NNAL and uCot varied according to the subjects'age.NNAL seems to play a role in decreasing sBP by enhancing uNa excretion.Insufficient nutrient intake may contribute to endogenous formation of NNAL in non-smoking adolescents and children.

IMMUNOCHEMICAL IDENTIFICATION AND LOCALIZATION OF CYTOCHROME P-450HSjISOZYME, AN ENZYME RELATED TO NITROSAMINE METABOLISM, IN HUMAN GASTRIC MUCOSA AND GASTRIC CARCINOMA

Monoclonal antibody (MAb) to rat liver cyto-chrome P-450j isozyme, an activating enzyme specific to nitrosamine metabolism, was used coupled with immunoblotting, densitometer scanning of SDS-PAGE gels and immunohistochemical technique. The trace P-450HSj isozyme (Mr. 51.5 Kd) was found in human gastric mucosa. It was similar to P-450j in molecular weight, catalytic and immunochemical properties. The concentrations of P-450HSj in mucosa of lesser curvature were higher than those in greater curvature. This might be one of the important reasons that lesser curvature is the commonest area for gastric carcinoma. But there was possibly less P-450HSj in gastric mucosa with cancer. Im-munohistochemically, P-450HSj was discovered in the cytoplasm of some glandular epithelial cells, especially in the glands with hyperplastic and intestinal metaplastic changes adjacent to carcinoma. It was also found in some normal glands and in tumor cells of high-differentiated adenocarcinoma, but not in those of low-differentiated ones. Following subjects are discussed: (1) the method of detecting trace P-450HSj, (2) the rule of distribution of P-450HSj, and (3) the relationship between the isozyme and the occurrence of gastric cancer caused by nitrosa-mines.

MECHANISM OF PROMOTING EFFECTS OF RIBOFLAVIN DEFICIENCY ON CARCINOGENESIS OF NITROSAMINES (EFFECTS ON RAT LIVER GLUTATHIONE CONTENT)

The effects of riboflavin deficiency and simultaneously nitrosodimethylamine (NDMA) given by gastric intubation on the hepatic glutathione (GSH) content were examined in rats. On different days of the experiment, hepatic GSH content of the riboflavin deficient rats decreased to 55-61% of the control rats. When NDMA was given 6 mg kg by gastric intubation to riboflavin deficient rats, hepatic GSH content decreased markedly to 39-43% of the control rats. After supplying riboflavin, hepatie GSH content of the deficient rats recovered to the level of the control rats. These results suggest that alterations of rat hepatic GSH content during riboflavin deficiency may imply as one of the promoting effects of riboflavin deficiency on the carcinogenesis of nitrosamines.

Analysis of Nitrosamines in Organic Foods

Nitrosamines are classified by IARC as Group 2B carcinogens. Usually they might be present in organic foods as products of reaction between secondary amines and nitrosation system. The aim of the study was to test the concentration of nitrosamines in Bulgarian products. High performance liquid chromatography with UV detector was used for identification and quantitation. A standard solution of N-nitrosodiethanolamine was used as a reference substance and in the validation procedure of samples. The limit of detection of the method was determined to 14× 10^-9 g/mL. The results of the testing showed that analyzed organic foods produced in Bulgaria did not contain nitrosamines above the limit of detection of the method.

Carcinogenesis by Nitrosamines and Azoxyalkanes by Different Routes of Administration to Rats

The effects of administering similar doses of some simple alkylating agents to rats by different regimens have been compared.Two of the compounds were methylating agents,nitrosodimeth- ylamine and azoxymethane;two were ethylating agents,nitrosodiethylamine and azoxyethane and nitrosomethylethylamine was both a methylating and an ethylating agent.The treatments gave rise to tumors in almost all treated rats.The results indicate the importance ofpharmacoki- netics in determining which organs are the targets of the alkylating carcinogens.1989 Academic Press.Inc.

Hemoglobin Adducts as Biomarkers of Exposure to and metabolic Activation of Carcinogenic Tobacco-Specific Nitrosamines

The carcinogenic tobacco-specific nitrosamines N-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK) form hemoglobin adducts in laboratory animals and humans. These adducts release 4-hydroxy-l-(3-pyridyl)-l-butanone (HPB) upon mild base hydrolysis. HPB released from human hemoglobin can be quantified by gas chromatography-mass spectrometry. It is the only available biochemical marker for determination of exposure to, and metabolic activation of, carcinogens present only in tobacco. Levels of HPB were highest in snuff-dippers, followed by smokers and nonsmokers. Large interindividual variations in adduct levels were observed. The relationship between HPB levels in globin and DNA of rats treated with NNK has been investigated in order to aid in interpretation of the data from humans. These studies have provided the initial database for understanding the metabolic activation of tobacco-specific nitrosamines in humans.

Intake of tobacco nitrosamines of smokers in various provinces of China and their cancer risk:A meta-analysis

Nitrosamines are a class of carcinogens which have been detected widely in food,water,some pharmaceuticals as well as tobacco.The objectives of this paper include reviewing the basic information on tobacco consumption and nitrosamine contents,and assessing the health risks of tobacco nitrosamines exposure to Chinese smokers.We searched the publications in English from“Web of Science”and those in Chinese from the“China National Knowledge Infrastructure”in 2022 and collected 151 literatures with valid information.The content of main nitrosamines in tobacco,including 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone(NNK),N-nitrosonornicotine(NNN),N-nitrosoanatabine(NAT),N-nitrosoanabasine(NAB),total tobacco-specific nitrosamines(TSNA),and N-nitrosodimethylamine(NDMA)were summarized.The information of daily tobacco consumption of smokers in 30 provinces of China was also collected.Then,the intakes of NNN,NNK,NAT,NAB,TSNAs,and NDMA via tobacco smoke were estimated as 1534ng/day,591 ng/day,685 ng/day,81 ng/day,2543 ng/day,and 484 ng/day by adult smokers in30 provinces,respectively.The cancer risk(CR)values for NNN and NNK inhalation intake were further calculated as 1.44×10^(-5)and 1.95×10^(-4).The CR value for NDMA intake via tobacco smoke(inhalation:1.66×10^(-4))indicates that NDMA is similarly dangerous in tobacco smoke when compared with the TSNAs.In China,the CR values caused by average nitrosamines intake via various exposures and their order can be estimated as the following:smoke(3.75×10^(-4))>food(1.74×10^(-4))>drinking water(1.38×10^(-5)).Smokers in China averagely suffer 200%of extra cancer risk caused by nitrosamines in tobacco when compared with non-smokers.

Occurrence and formation potential of nitrosamines in river water and ground water along the Songhua River, China

The presence of mutagenic and carcinogenic nitrosamines in water is of great concern. In this study, seven nitrosamines including N-nitrosodimethylamine （NDMA）, N-nitrosodiethylamine （NDEA）, N-nitrosomethylethylamine （NMEA）, N-nitrosopyrrolidine （NPyr）, N-nitrosopiperidine （NPip）, N-nitrosodi-n-propylamine （NDPA）, and N-nitrosodi-n-butyl-amine （NDBA） were investigated in river water and ground water samples collected from 5 representative cities （Jilin, Songyuan, Harbin, Jiamusi and Tongjiang） along the Songhua River. The total concentrations of nitrosamines in ground water were n.d. （not detected） to 60.8 ng/L, NDMA was the most frequently detected nitrosamines in ground water, followed by NDEA and NPip. Relatively high detected frequency and concentrations of NDMA were also observed in river water samples, and the total nitrosamines＇ concentration at midstream is always higher than that at upstream and downstream. After 24 hr chlorination, concentration of NDMA, NDBA was obviously increased but NDEA was reduced. Furthermore, UV2s4 showed a better relationship with NDMA-FP rather than dissolved organic carbon （DOC）. NI-h-N. and TDN.

Effect of activated sludge treatment on the formation of N–nitrosamines under different chloramination conditions

Municipal wastewater discharge is considered as one of the main sources of N-nitrosamine precursors which can impact the qualities of downstream source waters and reclaimed wastewaters for potable reuse. N –Nitrosamine precursors can be removed to various degrees during biological wastewater treatment(e.g., the activated sludge(AS) process). So far, little is known about the impact of the AS process on N-nitrosamine formation under practical disinfection condition(e.g., uniform formation condition(UFC)). In this study, N-nitrosamine UFC from selected model compounds, sewage components(i.e., blackwaters and greywaters) and sewage samples were comprehensively investigated during batch AS treatment tests. N –Nitrosodimethylamine(NDMA) formation from the tested precursor compounds(i.e., trimethylamine(TMA) and sumatriptan(SMTR)) under UFC chloramination decreased mostly after 6 or 24 hr treatment with different types of AS(i.e., domestic rural AS, domestic urban AS, and textile AS), and the reductions in NDMA UFC were comparable to their NDMA formation potential(FP) reductions. In urine and feces blackwaters, NDMA UFC increased after 6 or 24 hr treatment with the domestic(i.e., rural and urban) AS, while NDMA FP decreased substantially. The increases in NDMA UFC after AS treatment was presumably attributed to the removal of bulk organic matters(e.g., dissolved organic carbon(DOC)) which favored NDMA formation under UFC. On the other hand, in laundry greywaters having relatively abundant DOC, N-nitrosamine UFC was less affected by DOC removal before or after AS treatment, but decreased to similar degrees with N-nitrosamine FP. In sewage samples collected from wastewater treatment plants, N-nitrosamines UFC tended to increase or remain constant during AS treatment, despite the decreases in their FPs. These results suggest that biological wastewater treatment(e.g., the AS process) may not effectively reduce N-nitrosamine formation(e.g., measured under UFC) partially because the concurrent removal of bulk organic matters(e.g., DOC) favored N-nitrosamine formation in s econdary effluents.

Application of Sargassum fusiforme(Harv.)Setch.Extract in Cigarettes

[Objectives]This study was conducted to investigate the application of Sargassum fusiforme(Harv.)Setch.in cigarettes.[Methods]Tobacco-specific nitrosamines in the smoke of cigarettes added with ethanol extract of S.fusiforme were determined,and the compounds related to the aroma of S.fusiforme were identified by flavor-smelling experiment.[Results]With the addition of the ethanol extract of S.fusiforme,the decrease in the total amount of four tobacco-specific nitrosamines in mainstream cigarette smoke reached 16.42%.The results of the flavor-smelling experiment showed that the aroma of S.fusiforme might be related to(R)-5,6,7,7A-tetrahydro-4,4,7A-trimethyl-2(4H)-benzofuranone,glycerol,ethyl palmitate,methyl palmitate,ethyl linoleate,methyl(Z,Z,Z)-9,12,15-octadecatrienoate,ethyl(Z,Z,Z)-9,12,15-octadecatrienoate,phytol,and tetradecanoic acid.[Conclusions]The ethanol extract of S.fusiforme has the potential function of reducing the content of tobacco-specific nitrosamines in smoke and improving the taste of cigarettes.

Study on environmental etiology of high incidence areas of liver cancer in China

INTRODUCTIONChina is a country with a high incidence of liver cancer insome areas.Liver cancer has a wide distribution andthreatens human health seriously.A rough estimationshows that out of a population of 1.2×10~8 in liver

Risk assessment of chemical substances of safety concern generated in processed meats

This manuscript is reviewing the presence of substances of safety concern that may be generated in different types of meat products.Such substances include N-nitrosamines that can be generated under certain conditions when using nitrite as preservative,the polycyclic aromatic hydrocarbons generated as a consequence of particular smoking processes,heterocyclic aromatic amines generated under particular cooking conditions,compounds released from the oxidation of lipids and proteins,Maillard reaction products like acrylamide and carboxymethyl lysine,and amines that can be generated and accumulated in fermented meats.These hazardous compounds,their mechanisms of generation,risks for health and ways of preventing its presence in meat products are briefly described in this review.