EMA Q&A Appendix 1 update posted on Feb. 26, 2024

Limits of Nitrosamines
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An update of the document “Appendix 1: Acceptable intakes established for N-nitrosamines” has been posted on EMA website:

https://www.ema.europa.eu/en/documents/other/appendix-1-acceptable-intakes-established-n-nitrosamines_en.xlsx
Reference Number: EMA/72902/2024/Rev. 3

New:
2,2,5-trimethyl-3-nitroso-1,3-oxazolidine
N-nitroso-apixaban impurity B
N-nitroso-clozapine
N-nitroso-clozapine EP Impurity C
N-nitroso-desmethyl-tamoxifen
N-Nitrosodiisopropanolamine
N-nitroso-flecainide
N-nitroso-masitinib
N-nitroso-meglumine
N-nitroso-meropenem
N-nitroso-ritalinic acid
N-nitroso-silodosin
N-nitroso terazosin impurity C
N-nitroso terazosin impurity N

Update:
N-nitroso-folic acid

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Is tamoxifen not covered with ICH S9? I think it is used for treatment of advanced breast cancer. The AI of 18 ng/day looks so strict.

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In addition, in case useful :slight_smile:

x14 new
2,2,5-trimethyl-3-nitroso-1,3-oxazolidine (source Cyanocobalamine)  AI 1500 ng/day, CPCA Category 4
N-nitroso-apixaban impurity B (source Apixaban)  AI 1500 ng/day, CPCA Category 4
N-nitroso-clozapine (source Clozapine)  AI 1500 ng/day, CPCA Category 5
N-nitroso-clozapine EP Impurity C (source Clozapine)  AI 400 ng/day, CPCA Category 3
N-nitroso-desmethyl-tamoxifen (source Tamoxifen)  AI 18 ng/day, CPCA Category 1
N-Nitrosodiisopropanolamine (NDIPLA)  AI 400 ng/day, CPCa Category 3
N-nitroso-flecainide (source Flecainide)  AI 1500 ng/day, CPCA Category 4
N-nitroso-masitinib (source Masitinib)  AI 400 ng/day, CPCa Category 3
N-nitroso-meglumine (source Telmisartan)  AI 1500 ng/day, Limit based on negative bacterial reverse mutation test
N-nitroso-meropenem (source Meropenem)  AI 1500 ng/day, CPCA Category 4
N-nitroso-ritalinic acid (source Methylphenidate)  AI 1500 ng/day, CPCA Category 4
N-nitroso-silodosin (source Silodosin)  AI 1500 ng/day, CPCA Category 4
N-nitroso terazosin impurity C (source Terazosin)  AI 400 ng/day, CPCa Category 3
N-nitroso terazosin impurity N (source Terazosin)  AI 400 ng/day, CPCa Category 3

x1 updated:
N-nitroso-folic acid  AI 1500 ng/day, Limit based on negative bacterial reverse mutation test

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Hi Yosukemino,
yes I agree 18 ng/day is really low. From what I can see Tamoxifen is an hormonal treatment to be used alongside advanced cancer treatment; this may be why they consider it is not strictly within the S9 scope.

Saying this, I noticed in January that they had published an AI for Ribociclib, which is an advanced cancer treatment as per the published EPAR. The published category is 3 with AI 400 ng/day; but I thought at the time that publication of that AI was not in line with the EMA Q&A guideline.

I was wondering why an AI for Masitinib had been published this month by EMA ? I have checked the EMA website and Masitinib is authorised as a veterinary product to treat tumours in animals. It was refused human authorisation for the treatment of amyotrophic lateral sclerosis. Any thoughts?

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@stephanie.snow It looks like there are ongoing trials for Masitinib for covid and MS uses, amongst others - could be the reason that it has had a limit published.

It does raise the question around which compounds are having limits published nowadays. A lot of those in the original lists were for compounds that the EMA had been asked to product limits for at the start of the nitrosamines journey. Is the case now that the limits that are published are coming from submissions (clinical trials or product launches) from companies, or from company updates on levels detected at stage 2 and 3 of the nitrosamine process?

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Tamoxifen is clearly DNA-reactive and carcinogenic in rat liver. Since most human carcinogens are DNA-reactive carcinogens in rodents (20), such findings with tamoxifen raise grave concern. The observations that tamoxifen is also DNA-reactive in hamster liver and is activated by human liver microsomes to form DNA adducts provide evidence that its toxicity is not confined to the rat. Accordingly, tamoxifen must be presumed to be a human cancer hazard, unless some mechanistic basis for nonsusceptibility of humans is discovered. The presumption of hazard is reinforced by the finding of p53 mutations in the tamoxifen-induced liver tumors. The p53 gene is the most commonly mutated gene in human neoplasms (21, 22).

Source:Tamoxifen Experimental Carcinogenicity Studies: Implications for Human Effects

In my modest opinion, looking for nitrosamines in Tamoxifen does not make much sense.

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Hi Paliog, thanks a lot for sharing those.
This seems indeed strange since Q10 of the EMA Q&A version 20 also states that if the API is itself mutagenic or clastogenic at therapeutic concentrations, N-nitrosamine impurities should be controlled at limits for non-mutagenic impurities according to ICH M7(R2).

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based on experimental studies it has been confirmed formation of n-nitroso ticagrelor is not possible hence respective stakeholders are requested to look into. also requesting confirmation from the members/community to comment if anyone has structural confirmation and estimation

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Nitrosation reactions on Cyclopropyl amines where literature is saying that the Cyclopropyl amines are opening the rings while reacting with Nitrosating agent and forming stable five membered Isoxazoles like above i.e. cyclopropanes are converted to other derivative under acidic condition by N=O fragment insertion into three-carbon ring. it is confirmed that N-Nitroso Ticagrelor compound formation was not observed due to nitrosating reagent reacting with secondary amine of Ticagrelor and further converted to derivative Impurity under acidic condition.

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well pointed vinah for the fake ‘‘N-nitrosoticagrelor’’ and, of course, fully agree with your words.
It is weird why is still in the list, i could say weirder than its entrance :blush:

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Would it be possible that a cyanocobalamine medicine is the source but not the origin of this contamination (e.g. nitroso-meglumine type of reporting by the EMA)? I don’t see the immediate link with cyanocobalamine and 2,2,5-trimethyl-1,3-oxazolidine.

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In fact the relationship is not obvious at all.
2,2,5-trimethyl-1,3-oxazolidine is a possible by-product of a reagent used in the synthesis of Cyanocobalamine. According to the risk assessment of an API manufacturer, Isopropanolamine (prìmary amine) can react with nitrites, but the resulting diazonium ion is highly instable; the rearrangement of the carbocation may lead to a small amount of acetone; this reacts with the remaining Isopropanolamine to form 2,2,5-trimethyloxazolidine.
The latter is a secondary amine and may be nitrosated to form 2,2,5-trimethyl-3-nitroso-oxazolidine
image

Summarizing, this may be only a synthesis impurity of Cyanocobalamine; it cannot form (or increase) in the drug product.

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Have you checked the update to the AI list (Appendix 1: Acceptable Intakes Established for N-nitrosamines, Reference Number: EMA/72902/2024 Rev. 4)?

2-(4-nitrosopiperazin-1-yl)ethanol - 400 ng/dia (CPCA 3)

3-((ethyl(nitroso)amino)methyl) benzenesulfonate - 18 ng/dia (CPCA 1)

4-nitroso-methyl piperazine-1-carboxylate - 400 ng/dia (CPCA 3)

N-(2-hydroxy-2-phenylethyl)-N-(4-nitrophenethyl)nitrous amide - 400 ng/dia (CPCA 3)

N-(4-aminophenethyl)-N-(2-hydroxy-2-phenylethyl)nitrous amide - 400 ng/dia (CPCA 3)

N-nitroso-articaine - 1500 ng/dia (CPCA 4)

N-nitroso-desmethyl-edoxaban - 100 ng/dia (CPCA 2)

N-nitroso-fenfluramine - 400 ng/dia (CPCA 3)

N-nitroso-folic acid - 1500 ng/dia (CPCA 4)

N-nitroso-furosemide - 1500 ng/dia (Limit based on negative bacterial reverse mutation test)

N-nitroso-lisinopril – NMI (Limit derived using structure-activity-relationship and physicochemical features of N-nitroso-lisinopril, N-nitroso-quinapril and N-nitroso-quinaprilat)

N-nitroso-N-desmethyl-dextromethorphan - 1500 ng/dia (CPCA 5)

N-nitroso-N-ethyl-benzylamine - 18 ng/dia (CPCA 1)

N-nitroso-posaconazole Impurity 1 - 400 ng/dia (CPCA 3)

Rivaroxaban Nitroso Impurity 1 - 1500 ng/dia (CPCA 4)

Rivaroxaban Nitroso Impurity 2 - 1500 ng/dia (CPCA 4)

Rivaroxaban Nitroso Impurity 5 - 1500 ng/dia (CPCA 4)

N-nitroso-trientine Impurity 1 - 18 ng/dia (CPCA 1)

N-nitroso-vibegron - 1500 ng/dia (CPCA 5)

https://www.ema.europa.eu/en/documents/other/appendix-1-acceptable-intakes-established-n-nitrosamines_en.xlsx

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Dear @lucas10mauriz,

Thank you for the updates. I made a pie chart of CPCA classification in Appendix 1. There are still some category 1 and 2 compounds.

image

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I made a pie chart of “others”.

image

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Thanks @lucas10mauriz for sharing.

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I am interested in read-across for lisinopril to determine AI as NMI. Some of the N-nitroso-ACE inhibitors may be turned into NMI in the future as the EFPIA position paper mentioned.

image
lisinopril

image
quinapril

image
quinaprilat

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Dear Members,
I have noticed N-nitroso lisinopril declared as non-mutagenic solely based on read-across and physicochemical properties. This is little interesting to me. Do we have this type of case existed in prior - Where declaring a nitrosamine non-mutagenic based on Read-across and physicochemical properties.
If yes, please let me know or share here.

I would love to know what has triggered EMA to declare this. The only structural similarity is carbonyl moiety (C=0) attached to alpha carbon of N-nitroso moiety and and same carbonyl moiety (C=0) attached to hetro atoms (O&N).

I could not trace any data on N-nitroso Quniaprilat?

Please let me know if members have any other thoughts how this decision could have been arrived?

Molecular weight 434.49
Physical state Solid
pH Basic
Log P 1-2 (predicted)
Solubility Soluble in water & chloroform
GI absorption Expected to be low

image

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I’m wondering if calling this readacross has also procedural meaning.
Is this widening the option Q&A 10 B3 and thus meaning that such submissions by applicants go to NITWG for readacross alignment after NS OEG assessment?
Or is it still possible an option Q&A 10 B5 is added in the guidance (for justifiable class specific NMIs based on negative in vivo mutagenicity data for an analogue) or option Q&A 10 B4 is widened, possibly meaning no NITWG evaluation process (just NS OEG assessment) is required for such cases and thus a less strict interpretation of a need for a higher limit to allow case assessment?

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Dear @Yosukemino ,

Your graphical representation is quite good. Somehow, I can see that these percentages align with what has already been published in Revisiting the Landscape of Potential Small and Drug Substance Related Nitrosamines in Pharmaceuticals, as the authors state: 'Across each of the datasets analyzed, the number of theoretical nitrosamines assigned to categories 1 and 2 can be seen to represent a small fraction of the total number.

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