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).

Product Line-up

Calculation of Allowable Intake (AI) in the EMA CPCA

Flowchart to predict the Potency category of an N-nitrosamine

Does N-nitrosamine have any hydrogen on its α-carbons^*1?

Yes

Potency Category 5
AI = 1,500 ng/day

Does N-nitrosamine have more than one α-hydrogen^*1 on one or both sides of the N-nitroso group?

Yes

Potency Category 5
AI = 1,500 ng/day

Does N-nitrosamine have a tertiary α-carbon^*1?

Yes

Potency Category 5
AI = 1,500 ng/day

Calculate Potency Score^*2
Is score ≥ 4?

Yes

Potency Category 4
AI = 1,500 ng/day

Is Potency Score^*2 = 3?

Yes

Potency Category 3
AI = 400 ng/day

Is Potency Score^*2 = 2?

Yes

Potency Category 2
AI = 100 ng/day

Is Potency Score^*2 ≤ 1?

Yes

Potency Category 1
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*

* Sum all scores for features present in the N-nitrosamine

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

* A score of 3 applies when the methylene α-carbon is not part of an ethyl group. If the methylene α-carbon is part of an ethyl group, a score of 2 should be applied.

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

*1 Excludes examples where N-nitroso group is in a pyrrolidine ring, a 6-membered ring containing at least one sulfur atom or a morpholine ring (all counted separately).

*2 Excludes carboxylic acid and aryl (counted separately), and ketone (conflicting data). Additional electron withdrawing group examples are limited to those described in the following paper as “β-carbon electron withdrawing groups.”
Cross, K. P. and Ponting, D. J.: Comput Toxicol., 20, 100186 (2021).

*3 β-Carbon must be in an sp³ hybridisation state for this feature to apply.

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

Does N-nitrosamine have any hydrogen on its α-carbons?

Yes

Potency Category 5
AI = 1,500 ng/day

Does N-nitrosamine have more than one α-hydrogen on one or both sides of the N-nitroso group?

Yes

Potency Category 5
AI = 1,500 ng/day

Does N-nitrosamine have a tertiary α-carbon?

Yes

Potency Category 5
AI = 1,500 ng/day

Calculate Potency Score
Is score ≥ 4?

Yes

Potency Category 4
AI = 1,500 ng/day

Is Potency Score = 3?

Yes

Potency Category 3
AI = 400 ng/day

Is Potency Score = 2?

Yes

Potency Category 2
AI = 100 ng/day

Is Potency Score ≤ 1?

Yes

Potency Category 1
AI = 18 ng/day

For research use or further manufacturing use only. Not for use in diagnostic procedures.

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