Drug molecules have included stable heavy isotopes of carbon, hydrogen, and other elements, mostly as tracers for quantitation during the drug development process. Due to its potential to alter the pharmacokinetic and metabolic profiles of medications, deuteration has drawn attention[1].

Absorption, Distribution and Excretion
/Milk/ In goats, one hour after oral administration of 30 mg/kg body weight of N-nitrosodiethylamine, the N-nitrosodiethylamine content in milk was 11.4 mg/kg, and in blood it was 11.9 mg/kg. Trace amounts of N-nitrosodiethylamine were detected in milk after 24 hours, but not in blood. Autoradiography studies showed that unmetabolized N-nitrosodiethylamine was uniformly distributed in most fetal tissues on all study days of mouse pregnancy (days 12, 14, 16, and 18). Results also indicated that the substance was metabolized in the fetal bronchial tree mucosa and liver on day 18 of gestation.

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Metabolism/Metabolites The inhibition of sulfonyltransferases by 2,6-dichloro-4-nitrophenol completely eliminated the genotoxicity of N-nitrosodiethanolamine in rat liver, manifested as the induction of DNA single-strand breaks. N-Nitrosamino-2-hydroxymorpholine is an oxidative metabolite of N-nitrosodiethanolamine mediated by alcohol dehydrogenases, and its DNA strand-breaking ability is almost completely eliminated. Unlike these β-hydroxylated nitrosamines, 2,6-dichloro-4-nitrophenol has no effect on the DNA-damaging ability of N-nitrosodiethanolamine. This paper proposes a novel activation mechanism for N-nitrosodiethanolamine: N-nitrosodiethanolamine is first converted to a cyclic hemiacetal N-nitrosamino-2-hydroxymorpholine by alcohol dehydrogenases. This cyclic β-hydroxynitrosamine appears to be a substrate for sulfonyltransferases. The resulting sulfate conjugate is considered to be the final genotoxic electrophilic agent. However, the results do not rule out the possibility that N-nitrosodiethanolamine itself undergoes sulfate binding.
Oxidative N-deethylation of NDEA is the cause of CO2 and alkylation in vivo. We determined the metabolic rate of NDEA in rat and hamster organ sections in vitro and established the correlation between the degree of metabolism and induced tumor distribution. Following injection of NDEA into rats or hamsters, various ethylated derivatives were generated in the liver and kidney nucleic acids. These compounds included 7-ethylguanine, O6-ethylguanine, and 3-ethyladenine. ...Evidence suggests that nitrosodiethylamine requires metabolic activation to exert its carcinogenic and toxic effects. ...N-nitrosoethyl-N-(2-hydroxyethyl)amine and N-nitrosoethyl-N-(carboxymethyl)amine were detected in rat urine. ...The possible relationship between the structure and metabolism of nitrosamines has been investigated in the rat small intestine. Separated segments of the jejunum and ileum were perfused through the lumen for 2 hours. The perfusion fluid contained one of four symmetrical dialkylnitrosamines (each side chain containing 2-5 carbon atoms, all nitrosamines labeled with 14C at the α-position) or one of two asymmetrical nitrosamines (N-nitrosotert-butylmethylamine and N-nitrosomethylbenzylamine, labeled with 14C at the methyl group). In addition to determining the 14C content in the intestinal tissue, the presence of polar metabolites in the absorptive fluid (absorbate), perfusion fluid, and tissue homogenate was analyzed to assess the intestinal metabolism of nitrosamines. Neither N-nitrosodiethylamine nor the two asymmetrical nitrosamines underwent significant metabolism. Known metabolites of N-nitrosodiethylamine include N-nitrosoethylamine.

Toxicity Summary
Identification and Uses: N-nitrosodiethylamine (NDEA) is a yellow, oily substance. It is used as an additive in gasoline and lubricants, an antioxidant, and a plastic stabilizer. Human Studies: NDEA has been identified as a tobacco carcinogen. Short-term exposure of bronchial epithelial cell lines to tobacco carcinogens (including NDEA) equivalent to smoking concentrations altered the expression of key proliferation regulatory genes EGFR, BCL-2, BCL2L1, BIRC5, TP53, and MKI67, similar to changes reported in lung epithelial biopsy specimens described as precancerous lesions. Animal Studies: NDEA can induce tumors in various tissues and via various exposure routes in a variety of experimental animals. NDEA is carcinogenic in perinatal and adult animals, primarily leading to tumors of the liver, respiratory tract, kidneys, and upper gastrointestinal tract. Benign and malignant liver tumors have been observed in mice, rats, hamsters, guinea pigs, rabbits, dogs, and pigs after oral administration of NDEA. Liver tumors were induced in rats after inhalation or rectal administration; liver tumors were induced in mice, rats, and hamsters after intraperitoneal injection; liver tumors were induced in hamsters, guinea pigs, gerbils, and hedgehogs after subcutaneous injection; liver tumors were induced in mice after prenatal exposure; liver tumors were induced in birds after intramuscular injection; and liver tumors were induced in fish and frogs after exposure to NDEA in aquarium water. In dogs, NDEA administered via gastric tube followed by subcutaneous injection resulted in liver and nasal cancer. Lung and upper respiratory tract tumors were induced in mice, rats, hamsters, dogs, and pigs after oral administration of NDEA. Kidney tumors were induced in rats after oral, intravenous, or prenatal administration of NDEA. Oral administration of NDEA also caused kidney tumors in pigs and upper gastrointestinal tumors in mice, rats, and hamsters. The mutagenicity of NDEA was assessed using Salmonella test strains TA98, TA100, TA1535, TA1537, and TA1538 (Ames test) with and without metabolic activation. Regardless of metabolic activation, NDEA did not induce reproducible positive reactions in any of the tested bacterial strains. In the presence of phenobarbital-treated rat liver microsomal fractions, NDEA induced 8-azaguanine-resistant mutants in Chinese hamster V79 cells. NDEA exhibited mutagenicity in a recessive lethal assay in Drosophila. This study investigated the effects of NDEA on sexual development, gametogenesis, and oocyte maturation in Japanese medaka (Oryzias latipes). Results showed that NDEA dose-dependently reduced germ cell number in early sexual differentiation of XX juveniles, leading to incomplete ovarian development in adults. This effect was sex-specific; no similar changes were observed in XY juveniles. Furthermore, XX and XY juveniles exposed to low doses of NDEA early on showed significantly reduced adult body weight. Compared to the control group, sexually immature adult males and females exposed to NDEA exhibited more advanced gonadal development. Ecotoxicity studies showed that NDEA concentrations exceeding 5 μg/L induced oxidative stress and antioxidant defense responses in the zebrafish metabolic system. Significant DNA damage was observed in zebrafish hepatocytes when NDEA concentrations exceeded 500 μg/L after 42 days of exposure. Toxicity and carcinogenicity were observed in Phython reticulatus after lifelong gavage administration of 6, 12, and 24 mg/kg NDEA (once nightly for four times). The total dose required to induce tumors was 500–600 mg/kg. Interactions Objective: This study aimed to investigate the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and N-nitrosodiethylamine (DEN) on tumorigenesis and their potential mechanisms. Methods: The potential for TCDD and DEN to induce malignant transformation, alone or in combination, was tested in Balb/c 3T3 cells using a cell transformation assay. The possible mechanisms of the observed effects were further investigated by adding α-naphthylflavonoid (α-NF), a competitive binder of TCDD, to the aryl hydrocarbon receptor (AhR) pathway. The mRNA expression levels of Cyp1a1 and Cyp2a5 genes in Balb/c 3T3 cells were detected by quantitative real-time RT-PCR using DEN and TCDD alone or in combination, with or without α-NF in the combination treatment group. Results showed that the cell transformation frequency (TF) induced by TCDD combined with DEN was significantly increased compared to TCDD or DEN alone. α-NF did not inhibit this effect. TCDD alone enhanced the mRNA expression levels of Cyp1a1 and Cyp2a5, but the combination of TCDD and DEN blocked this induction effect. Conclusion: The combination of TCDD and DEN has a significant synergistic effect on tumorigenesis. The AhR pathway may not be the key mechanism of this synergistic effect. Therefore, further investigation of the potential mechanisms involved in cancer development is necessary. ...In this study, we investigated the effect of pitavastatin, a drug used to treat hyperlipidemia, on the development of diethylnitrosamine (DEN)-induced precancerous lesions in the liver of C57BL/KsJ-db/db (db/db) obese mice. Male db/db mice were given tap water containing 40 ppm DEN for 2 weeks, followed by diets containing 1 ppm or 10 ppm pitavastatin for 14 weeks. After sacrifice, compared with the untreated group, administration of 10 ppm pitavastatin significantly inhibited the development of precancerous lesions (cellular alterations) in the liver, through inducing apoptosis rather than inhibiting cell proliferation. Pitavastatin improved hepatic steatosis and activated AMPK-α protein in the liver. It also reduced serum levels of free fatty acids and aminotransferases while increasing adiponectin levels. Pitavastatin treatment reduced serum levels of tumor necrosis factor (TNF)-α and the expression of TNF-α and interleukin-6 mRNA in the liver, indicating that it alleviates chronic inflammation caused by excessive fat deposition. Resveratrol is a phytochemical abundant in red wine and grapes, known to affect cancer cells both in vitro and in vivo. This study reports the effects of resveratrol on early and late stages of hepatocellular carcinoma (HCC) in a male Wistar rat model induced by N-nitrosodiethylamine (DEN). Rats were divided into two groups and treated with resveratrol for 15 days starting on day 1 after DEN administration (before HCC) or 15–16 weeks after HCC development (post-HCC). These groups were compared with untreated HCC rats. Biochemical analysis of known serum markers of HCC, such as alpha-fetoprotein (AFP), and other serum and liver marker enzymes showed that these levels were lower in the resveratrol-treated groups compared to untreated HCC rats. Hematoxylin-eosin staining of liver tissue sections revealed alterations or transformations in the liver parenchyma during DEN-induced HCC (15–16 weeks). Resveratrol treatment in early (day 1 after DEN induction) and late (weeks 17-18) HCC resulted in significant differences in tissue structure compared to untreated HCC. Immunoblotting analysis showed that resveratrol intervention activated apoptosis markers, such as PARP lysis, caspase-3 activation, p53 upregulation, and cytochrome c release, in both early and late stages of DEN-induced hepatocellular carcinoma (HCC). Furthermore, semi-quantitative RT-PCR and immunoblotting analysis indicated that the expression of key apoptosis regulators Bax and Bcl2 was upregulated and downregulated, respectively, in a resveratrol-dependent manner. ...
This study evaluated the chemopreventive potential of purple bean extract (TPE) against N-nitrosodiethylamine (NDEA)-induced hepatocellular carcinoma (HCC) in Wistar rats. HCC was induced by intraperitoneal injection of NDEA (200 mg/kg), followed by weekly subcutaneous injections of CCl₄ (3 mL/kg) for six weeks. Following administration of the carcinogen, this study administered TPE orally once daily at doses of 200 and 400 mg/kg. NDEA treatment significantly increased the levels of hepatocellular carcinoma markers, including alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA). TPE treatment significantly reduced liver injury and normalized all hepatocellular carcinoma markers. Furthermore, TPE significantly restored the activity of antioxidant enzymes in the liver of NDEA-treated rats, including lipid peroxidase, reduced glutathione, catalase, superoxide dismutase, glutathione peroxidase, and glutathione S-transferase. TPE treatment significantly reduced the incidence and number of carcinogen-induced hepatic nodules in rats. Histological findings were consistent with biochemical findings. These findings strongly support the role of T. purpurea in inhibiting lipid peroxidation, suppressing tumor burden, and promoting enzymatic and non-enzymatic antioxidant defense systems during NDEA-induced hepatocellular carcinogenesis. This may be due to its modulation of antioxidant defense status, thereby enhancing its anticancer potential. For more complete data on interactions of N-nitrosodiethylamine (16 in total), please visit the HSDB record page.

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Non-human toxicity values
Subcutaneous LD50 in rats: 195 mg/kg
Intraperitoneal LD50 in rats: 216 mg/kg
Intravenous LD50 in rats: 280 mg/kg
Oral LD50 in rats: 280 mg/kg
For more complete data on non-human toxicity of N-nitrosodiethylamine (6 in total), please visit the HSDB record page.

[1]. Impact of Deuterium Substitution on the Pharmacokinetics of Pharmaceuticals. Ann Pharmacother. 2019 Feb;53(2):211-216.C

According to an independent committee of scientific and health experts, N-nitrosodiethylamine is potentially carcinogenic. N-nitrosodiethylamine is a clear, pale yellow liquid with a boiling point of 175-177°C. It is reasonable to expect it to be a carcinogen. It is used as an additive in gasoline and lubricants, as well as an antioxidant and stabilizer in plastics. N-nitrosodiethylamine is a nitrosamine, a product of N-ethylethylamine with a nitroso group replacing the nitrogen atom. It is mutagenic, hepatotoxic, and carcinogenic. N-nitrosodiethylamine is a synthetic, photosensitive, volatile, clear yellow oily substance, soluble in water, lipids, and other organic solvents. It is used as an additive in gasoline and lubricants, an antioxidant, and a stabilizer in industrial materials. Upon heating and decomposition, N-nitrosodiethylamine releases toxic nitrogen oxide fumes. N-nitrosodiethylamine may affect DNA integrity through alkylation and has been used in experimental studies to induce liver tumors. It is considered a likely human carcinogen. (NCI05)
A nitrosamine derivative with alkylating, carcinogenic, and mutagenic properties.

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Mechanism of Action
…Studies have shown that two nitrosamines, N-nitrosodiethylamine and 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone, can bind to nicotinic cholinergic receptors in hamster lung. The binding of nitrosamines and nicotine to these receptors can stimulate the proliferation of human lung carcinoid cancer cells in vitro. These data suggest that chronic stimulation of nicotine receptors by nicotine and nitrosamines in smokers is one of the molecular mechanisms leading to the proliferation of neuroendocrine cells and ultimately the development of lung tumors with neuroendocrine differentiation characteristics.

C4H6D4N2O

106.16

106.104

1346603-41-5

N-Nitrosodiethylamine;55-18-5

5921

Yellow oil
Slightly yellow liquid

0.9±0.1 g/cm3

173.9±9.0 °C at 760 mmHg

59.0±18.7 °C

1.7±0.3 mmHg at 25°C

1.442

0.42

0

3

2

7

51.7

0

CCN(CC)N=O

WBNQDOYYEUMPFS-VEPVEJTGSA-N

InChI=1S/C4H10N2O/c1-3-6(4-2)5-7/h3-4H2,1-2H3/i1D2,3D2

N-ethyl-N-(1,1,2,2-tetradeuterioethyl)nitrous amide

DEN-d4; Diethylnitrosamine-d4; N-Nitrosodiethylamine-d4

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)