N-nitrosamine risk assessment in pharmaceuticals: Where are we from a regulatory point of view in 2025?
Zhang2025 (Nitrosamines).pdf (2.3 MB)
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Very informative! I have a query regarding the statement mentioned below. Could someone please shed some light on this?
“Regulatory authorities have started recommending NAP tests to assess the risk of NDSRI formation from APIs. It is advised that the NAP test be performed on starting materials, intermediates, and APIs during manufacturing process development”
Reference page # 10/15
Do we need to perform NAP test even if there is no amine functional group present in the respective SM/ Intermediate/ API and neither does it have any precursor of amines in the entire process?
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Dear Gautam,
if no amine fuctional group is presented then no risk of NDSRIs is needed to be assessed, so there is no need to perform any NAP test.
thanx,
Christos
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Thank you so much Christos!!!
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Agreed. The NAP test is primarily used when you identify a vulnerable amine but have some indication that formation will not occur. My rule of thumb for generics is simply whether or not I can buy a reference standard of the NDSRI from a source I trust. If I cannot purchase a standard, I presume that the NDSRI does not form & we perform the NAP test. For NCE’s, we recommend always performing the NAP test for a vulnerable amine. I would also say that we always use the modified NAP test, using t-butyl nitrite in place of sodium nitrite.
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Posting this here in case it may be useful. We have been following the procedure outlined in this paper; Modified NAP test: A simple and Responsive Nitrosating Methodology for Risk Evaluation of NDSRIs - ScienceDirect. We have found that the samples are much cleaner than the original NAP test. Additionally, you are more likely to achieve full nitrosation. We typically use all of the solvents listed, ACN, ACN:H2O, and ACH:1M HCl.
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No, if there is no secondary, tertiary amine or an amide of a secodary amine, NAP test is of no use. WHO NAP test is when you put your compound in a nitrite and acid (Acetic or HCl) at a pH of 3-4 and stirr it for hours to see if a nitrosamine is formed. Again, the solubility of you compound in the media you use is important. So, if the compound in question does not dissolve in the medium, test may not be valid. You may have to also look into nitrosating agents like isopropylnitrite, nitrosotetrafluroborate. Lets not forget that NAP test was proposed in 1970s and is rudimentary. The joke is that as a former FDA I can tell you that in the early 80s, the FDA used to ask sponsors to do NAP test for risky compounds. I have seen several NDAs where NAP or similar tests were performed. But I think it went nowhere at that time and the trend died. And here we are in 2025 going back to a 1970s and reinventing the wheel. 
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