N-Nitrosamines: An Enhanced Ames Test with Hamster liver S9
Keywords: N-Nitrosamines, NDSRIs, OECD 471, Enhanced Ames test, Hamster liver S9, Cytochrome P450 enzymes, Mutation Test
IPHASE Products
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Product Name |
Specification |
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IPHASE Hamster (Golden Syrian) Liver S9,Induction |
35mg/Ml, 1mL |
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IPHASE Rat(Sprague-Dawley) Liver S9,Induction |
35mg/Ml, 1mL |
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IPHASE In Vitro Mammalian Cell Micronucleus Test |
5mL*32 test |
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IPHASE Ames Test Kit |
100/150/200/250 dishes |
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IPHASE Mini-Ames Test Kit |
6well*24/ 6well*40 |
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IPHASE Microtitre Fluctuation Ames Test Kit |
16*96 wells/ 4*384 wells |
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IPHASE UMU Genotoxicity Test Kit |
96 well |
|
Cell Gene Mutation Test (TK) Kit |
20ml*36 test |
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Cell Gene Mutation Test (HGPRT) Kit |
20ml*36 test |
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In-Vitro Chromosome Aberration Test Kit |
5mL*30 test |
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IPHASE Comet Assay Kit |
20/50 test |
N-Nitrosamines Impurities
N-nitrosamines (NDSRIs), with the structural formula R1(R2)N-N=O, are produced by various mechanisms, such as the reaction between secondary amines and nitrous acid, tertiary amines and monochloramine, N-dimethylformamide (DMF) oxidation, and the action of rubber vulcanizing agents and nitrogen oxides, etc. They are widely found in nature and food, such as air, water, soil, bacon, alcoholic beverages, etc. They may also be generated during drug production. NDSRIs are genotoxic, causing DNA damage and cancer, and their risk is not related to the dose, and very low doses can cause damage. The International Agency for Research on Cancer (IARC) has classified a variety of N-nitrosamines as carcinogens, with N-dimethylnitrosamine (NDMA) and N-diethylnitrosamine (NDEA) in category 2A and others in category 2B. The International Council for Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) M7 guideline classifies high carcinogenicity risk NDSRIs as Class I impurities, which need to be strictly controlled. The carcinogenicity of NDSRIs originates from their metabolites, and according to the ɑ-hydroxylation hypothesis, activation of NDSRIs by hydroxylation enzymes results in the formation of carcinogenic intermediates, which lead to alkylation of DNA bases and the induction of cancer.
Fig.1. Mechanism of NDMA's carcinogenicity
Enhanced Ames test on the Genotoxicity Assay of N-Nitrosamines Impurities
Since its inception in 1975, the Ames test, also known as the bacterial revertant mutation assay, has emerged as a fundamental tool in genotoxicological assessments for the preliminary screening of compounds to evaluate their mutagenic and potential carcinogenic properties. According to the latest guidance issued by the European Medicines Agency (EMA), the conventional Ames test may not be sufficiently sensitive to detect the mutagenic potential of certain N-nitrosamine impurities, particularly those such as N-Nitrosodimethylamine (NDMA). Therefore, the Enhanced Ames test, developed by the National Center for Toxicological Research (NCTR) under the U.S. Food and Drug Administration (FDA), is recommended as a more robust alternative. If the standard Ames test yields a positive result, the Enhanced version is not required; however, if the result is negative, further evaluation using the Enhanced Ames test is mandated. Should the Enhanced test also return a negative result, an in vivo mutagenicity assay becomes necessary, as the carcinogenic potential of N-nitrosamines cannot be comprehensively assessed through in vitro testing alone. In cases where the in vivo test is negative, an expert review is essential to interpret the findings. It is important to note that certain N-nitrosamine impurities may exhibit non-mutagenic properties while retaining carcinogenic potential, necessitating careful consideration and specialized attention in risk assessment.
The following Enhanced Ames Test (EAT) conditions are provided by the FDA
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Test strains |
Includes Salmonella typhimurium TA98, TA100. TA1535,TA1537 and Escherichia coli WP2 uvrA (pKM101) test strain |
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Test method and pre-insulation time |
Pre-insulation and non-flatbedding methods should be used, with a recommended pre-insulation time of 30 minutes. |
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S9 Type and Concentration |
The Enhanced Ames test should be performed in containing 30% rat liver S9 and 30% Hamster Liver S9. Rat and hamster desmosomal supernatants (S9s) should be prepared from rodent livers treated with cytochrome P450 enzyme-inducing substances (e.g., a combination of phenobarbital and β-naphthoflavone). |
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Negative (solvent/excipient) control |
The solvents used should be compatible with the Ames test according to OECD 471 guidelines. Available solvents include, but are not limited to: (1) Water; (2) Organic solvents such as acetonitrile, methanol, and dimethyl sulfoxide (DMSO). When organic solvents are used, the lowest possible volume in the pre-holding mixture should be used and it should be demonstrated that the amount of organic solvent used does not interfere with the metabolic activation of N-nitrosamines. |
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Positive Control |
According to OECD 471 guidelines, strain-specific positive controls should be performed at the same time. In the presence of S9, the two N-nitrosamines known to be mutagenic should also be used as positive controls. Available N-nitrosamine positive controls include: NDMA, 1-cyclopentyl-4-nitrosopiperazine NDSRIs。 |
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All other recommendations on Ames determination should follow OECD 471 guidelines |
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IPHASE Related Products
IPHASE, as a leader in biological reagents for in vitro research, with advanced equipment, professional technicians and years of experience in research and development, following the successful development of induced SD rat liver S9, we have successfully developed the first induced Hamster (Golden Syrian) liver S9 (Hamster Liver S9). The product was prepared through the combined induction of sodium phenobarbital and β-naphthoflavone. After aseptic inspection, metabolic activity test and quality control of Ames and chromosome aberration tests, the results showed that its performance was close to that of induced SD rat liver S9, and it complied with the standard of in vitro genotoxicity test.
In addition, IPHASE has now developed a series of products specializing in in vitro genotoxicity research, such as Genotoxicity Ames Kit and In Vitro Chromosome Aberration Kit, to help our customers conduct in vitro genotoxicology research.
About IPHASE
With years of R&D experience, IPHASE has launched high-end scientific research reagents in many fields and types, providing screening tools for early drug development, new materials, new methods and new means for the exploration of the field of life sciences, and convenient products for genotoxicity research of food, pharmaceuticals, chemicals, etc.
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