Nitrosamines Test
Nitrosamines are a group of compounds with a structure in which a hydrogen bound to the amine nitrogen is replaced by a nitroso group, and some of these compounds are known to be carcinogenic.
Nitrosamines are also formed as a reaction product of secondary amines and nitrous acid, so they can be detected as impurities in the manufacturing process of pharmaceuticals and are controlled under ICH M7 (Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk). In recent years, there have been cases of nitrosamines being detected in sartan and latinidine drugs, resulting in recalls. In response, in September 2019, the EMA (European Medicines Agency) requested the marketing authorization holders to evaluate the risk of the presence of nitrosamine impurities in their drugs and take appropriate risk mitigation measures.
Recently, there is also concern about the detection of nitrosamines for which there is no carcinogenicity data, as well as nitrosamine drug substance related impurities (NDSRI). In response, on July 7, 2023, the EMA's guideline for nitrosamines EMA/409815/2020 was revised and a new evaluation method, the Carcinogenic Potency Categorization Approach (CPCA). The CPCA is a method to calculate the allowable intake (AI) of nitrosamines by classifying them into five Potency categories based on the number of hydrogen atoms bound to the α-carbon of the N-nitroso group and their structural features, assuming that the carcinogenicity of nitrosamines is caused by the carbon at the α-position of the nitroso group.
Fujifilm Wako offers a wide range of analytical standards and mixture standard solutions for nitrosamines of various structures*1.
*1 This product is not an analytical standard (JP, EP, USP).
Calculation of Allowable Intake (AI) in the EMA CPCA
Flowchart to predict the Potency category of an N-nitrosamine
AI = 1,500 ng/day
AI = 1,500 ng/day
AI = 1,500 ng/day
Is score ≥ 4?
AI = 1,500 ng/day
AI = 400 ng/day
AI = 100 ng/day
AI = 18 ng/day
*1 1st carbon bound to N-nitroso group
*2 See below
Calculation of Potency score
Potency score = α-Hydrogen score + Deactivating feature score* + Activating feature score*
α-Hydrogen score
Count of hydrogen atoms on each α-carbon, lowest first | α-Hydrogen score |
Structural formula example |
---|---|---|
0, 2 | 3* | |
0, 3 | 2 | |
1, 2 | 3 | |
1, 3 | 3 | |
2, 2 | 1 | |
2, 3 | 1 |
Deactivating feature score
Deactivating features | Individual deactivating feature score |
Structural formula example |
---|---|---|
Carboxylic acid group anywhere on molecule | +3 | |
N-Nitroso group in a pyrrolidine ring | +3 | |
N-Nitroso group in a 6-membered ring containing at least one sulfur atom | +3 | |
N-Nitroso group in a 5- or 6-membered ring*1 | +2 | |
N-Nitroso group in a morpholine ring | +1 | |
N-Nitroso group in a 7-membered ring | +1 | |
Chains of ≥ 5 consecutive non-hydrogen atoms (cyclic or acyclic) on both side of acyclic N-nitroso group. Not more than 4 atoms in each chain may be in the same ring. | +1 | |
Electron-withdrawing groups*2 bonded to α-carbon on only one side of N-nitroso group (cyclic or acyclic) | +1 | |
Electron-withdrawing groups*2 bonded to α-carbons on both sides of N-nitroso group (cyclic or acyclic) | +2 | |
Hydroxyl group bonded to β-carbon*3 on only one side of N-nitroso group (cyclic or acyclic) | +1 | |
Hydroxyl group bonded to β-carbon*3 on both sides of N-nitroso group (cyclic or acyclic) | +2 |
Cross, K. P. and Ponting, D. J.: Comput Toxicol., 20, 100186 (2021).
Activating feature score
Activating features | Individual activating feature score |
Structural formula example |
---|---|---|
Aryl group bonded to α-carbon (i.e., benzylic or pseudo-benzylic substituent on N-nitroso group) | -1 | |
Methyl group bonded to β-carbon (cyclic or acyclic) | -1 |
Example of calculating AI of N-Nitrosolorcaserin using CPCA
Since N-Nitrosolorcaserin has multiple hydrogens on the α-carbons on both sides of the N-nitroso group (not tertiary), it is necessary to calculate a Potency score. The Potency score was calculated as follows: Potency score = 1, which classifies it as Potency category 1, and the AI = 18 ng/day.
α-Hydrogen score
Count of hydrogen atoms on each α-carbon, lowest first | α-Hydrogen score |
Applicable structural formula |
---|---|---|
2, 2 | 1 |
Deactivating feature score
Deactivating feature | Individual deactivating feature Score |
Applicable structural formula |
---|---|---|
N-Nitroso group in a 7-membered ring | +1 |
Activating feature score
Activating feature | Individual activating feature score |
Applicable structural formula |
---|---|---|
Methyl group bonded to β-carbon (cyclic or acyclic) | -1 |
Potency score = 1 + 1 - 1 = 1
AI = 1,500 ng/day
AI = 1,500 ng/day
AI = 1,500 ng/day
Is score ≥ 4?
AI = 1,500 ng/day
AI = 400 ng/day
AI = 100 ng/day
AI = 18 ng/day
For research use or further manufacturing use only. Not for use in diagnostic procedures.
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