Considering the discussion on NDSRIs, I’d like to raise a question regarding the updated version of the “NAP test”.
This test serves as a valuable tool in assessing the potential formation of nitrosamines in pharmaceutical products. By utilizing in vitro forced degradation conditions with an excess of nitrite in an acidic solution, the NAP test aims to simulate the in vivo formation of nitrosamines in the stomach.
Originally designed in 1980 by the International Agency for Research on Cancer (IARC) of the World Health Organization (WHO), the NAP test was not included in registration dossiers due to the limited availability of selective and sensitive analytical techniques at that time. However, its significance has grown with the advent of advanced analytical methods capable of detecting and quantifying nitrosamines (Arch. Pharm. 2022;355:e2100435).
The NAP test results provide valuable insights by excluding the formation of theoretically proposed nitrosamines derived from the chemical structure of the API or excipient. If the predicted nitrosamine structure cannot be detected after incubation under the specified conditions, it becomes highly unlikely that it will form in the final product.
In contrast to the WHO NAP test, which investigates potential in vivo N-nitrosamine formation using a large excess of nitrite, elevated temperatures, and only inorganic nitrite, the proposed reaction conditions for the updated version are more relevant to NDSRI formation in drug substance and product manufacturing processes. These conditions involve (doi.org/10.1021/acs.oprd.3c00084):
The use of an excess of nitrosating agent (1.5 molar equivalents),
Orthogonal approaches (utilizing both inorganic and organic nitrite, considering the presence or absence of chloride ions),
Room temperature to prevent nitrite decomposition.
Now, considering this information, I’d like to pose the following question: Are you familiar with this updated version of the “NAP test” for assessing the potential formation of NDSRIs in pharmaceutical products?
I’d appreciate your insights and thoughts on the relevance and applicability of such testing methodologies in ensuring product safety and quality.
Please feel free to share your perspectives and engage in this important discussion.
Thank you for the very interesint update, i was not aware of.
What i know is that the old test was discussed in several symposiums and i understood that one part was highlighting that risk is very high to form nitrosamines which can not be formed under manufacturing or product storage conditions, it seems this is now aligned. I know from som other dicussions that 1 or 2 companies in germany have been using widely this test and were challenged by a high number of possible nitrosamines to evaluate.
On question regarding the doing. you check per NMR or MS if a new peak is present and afterwards you would try to buy/synthesize the respective standards of the proposed nitrosamine for verification?
A methodology such as NAP can indeed predict the formation of nitrosamines, as I previously mentioned. However, in practical terms, it is possible that the specific nitrosamine may not be formed due to factors such as high reactivity and/or instability. Consequently, there may be practical limitations in isolating and analyzing it using techniques like Mass Spectrometry (MS) or Nuclear Magnetic Resonance (NMR).
That option has been explored in several cases with additional supporting data, but it’s not clear from the regulatory guidances to fully support derisk a nitrosamines impurity from additional assessment.
Related to this, please look at the presentation that Andrew Teasdale did at the recent CRGC meeting ‘Hydrochlorothiazide – a twist in the tail’
To substantiate the significance of the NAP test, Schmidtsdorff et al. (2022) unveiled compelling findings: out of a total of 67 drug substances investigated, 33 of them demonstrated to form NDSRIs in considerable quantities under NAP test conditions, as evidenced by SFC-MS/MS analysis. This observation suggests the potential application of the NAP test for evaluating the propensity of drug compounds to undergo nitrosation.
Regional guidance, as outlined below, places emphasis on the necessity for manufacturers to undertake risk assessments and confirmatory testing. These evaluations serve the purpose of providing valuable insights into the nitrosatability of amines present in medicinal products.
To my knowledge, there has been a longstanding absence of guidance when it comes to examining nitrosatability and endogenous nitrosation during the process of clinical development. But, given the evolving regulatory landscape, I believe it is essential to critically evaluate the suitability of the NAP test, Do you agree?
While i agree that NAP helps in predicting the formation of NDSRI in drug substances , the following design could be more appropriate for drug products.
Mix Nitrate/Nitrite salts to API, Placebo and to a mixture placebo and API / drug product powder .This is to mimic the actual composition of drug product
2)Exposing above samples to Heat ,Humidity and light .
3)Analysis and interpretation.