A limit of 96 is almost certainly derived by read-across from NDMA, which can be at least somewhat justified for any N-methyl-N-nitroso group (though there’s normally a more precise analogue than this). Remember that the FDA have not (yet? my understanding is that there has been regulatory collaboration on this so we might see changes in future) published the CPCA, so the approach is currently formally only valid for EMA submissions and any health authority that has specifically written their guidance as “copy EMA exactly”.
The way the EMA guidance is written, the CPCA should be performed first, but can be over-ruled with robust data, either a robust read-across or experimental toxicity data.
That’s really interesting explanation! Don’t you think that a glossary or definition list should accompany such guidelines to ensure eliminating any misunderstanding or misinterpretation?
I think “aryl” and “aromatic” are generally understood as described - especially when looking at the examples in the guidance and the limits for those compounds that have CPCA limits… in terms of a specific glossary, I suspect we’re out of luck due to the decision to refer the electron-withdrawing group (EWG) definitions back to Cross & Ponting rather than enumerating them.
My general thought would be to ask here (see for example the discussions of beta-methyl groups in another thread) if there are any features that the precise definitions of which are unclear - for example, I can state with some confidence that the EWG list, though there are many other groups that do withdraw electrons, is pattern-able as “*A=,#Q, *CF3, not *C([C,H])=O and not *COOH”.
that is I believe the structure - it’s not nitroso-sitagliptin itself, rather the nitrosamine of an impurity of sitagliptin.
It has been assigned a compound-specific limit of 37 ng/day by read-across from nitroso-1,2,3,6-tetrahydropyridine (with the aromatic group assumed to be comparable to the double bond there and the ‘arylic’ position like the allylic in NTHP), though by CPCA would have a limit of 100:
(2,2) for H-count = 1
In a generic 6-membered ring = 2
Aromatic group at b-position activating it = -1
Total 2, limit 100 ng/day
@David Thanks for clarity on subject.
However still question is that, which approach for determination AI is suitable and valid, either SAR or CPCA and how it will be decided.
My understanding is that in this case, because there is a defined limit, that takes precedence, however in the general case CPCA should be tried first but can be overridden if needed (and it will be - there are a lot of nitrosamines still in categories 1-3!) by read-across.
And David, do you think that the double bond is a really suitable analogue for the aromatic ring? The CF3 is a strong electron withdrawing group that can certainly render the aromatic ring with very unique electronic properties that would be have an impact on the alpha hydrogen of the nitrosamine, and it would be very different from the double bond.
In fact, while it cannot be categorized currently as an EWG, it may have that effect. Do you think that modelling softwares could predict if this would be the case?
Dear @ASrinivasan, thank you very much for the reply!
How do you see this issue: Additionally, the potency categorization approach does not apply to Nitrosamines where the N-nitroso group is within an aromatic ring (e.g., nitrosated indole).
Is the CPCA not applicable here and we have to use 18ng/day as AI, or are nitrosamines based on these structures not to be considered at all? This would make life quite easy for all potential imidazole-based nitrosamines.
For the nitrosated indoles, they aren’t nitrosamines per se; the indole nitrogen is not a secondary amine. Definitely still worth mentioning in the assessment if you consider there is risk of formation or have evidence thereof (don’t conceal anything is normally good advice!), but the implausibility of diazonium formation means that I would argue very strongly for treating these as conventional impurities under ICH M7 rather than as part of the cohort of concern.
We are asked to test 3-methyl-1-nitrosoindoline, 1-nitroso-indoline and 2-methyl-1-nitroso-indole as a part of nitrosamine risk assessment by Health Canada in recent query.
Modelling software certainly could have an impact - it’s entirely plausible that an electron-poor ring like this is going to be electron-withdrawing, at which point it could be argued that the +1 and -1 would cancel each other out. However, the CPCA is designed to be facile to implement, and conservative, so including this evidence moves out of the CPCA to a weight-of-evidence assessment.
As a result it would need a substantive research project and sufficient evidence to argue this to the point it could be included in a revised CPCA.
Only one of those is a nitrosated aromatic system - the two indolines both have the nitrosamine in non-aromatic rings (though fused to aromatic, yes) and the nitrogen has true single bonds.
1-nitrosoindoline would be category 4 by CPCA:
(0,2) for hydrogen count, and in a generic 5-membered ring, for a total of 5 points.
3-methyl-1-nitrosoindoline would be category 4 as well, with the same features but also the beta-methyl for an activating feature and a total of 4 points
I’m unsure why you were asked to test 2-Methyl-1-Nitroso indole (in an aromatic system and completely lacks a-hydrogens!), but would be really interested to see the data if you do - I’d expect a very different Ames strain profile (TA98/100 +/- S9, as described in Ponting and Foster).
Hi david,
you have applied CPCA approach for the “1-nitrosoindoline” (See below the structure) and “3-methyl-1-nitrosoindoline” (See below the structure) as they are considered as non-aromatic ring. and both are falls under category-4 and AI limit would be 1500 ng/day.
But, “2-methyl-1-nitrosoindoline” (See below the structure), which is present as an impurity in the Indapamide API EP monograph with a limit of 5 ppm and corresponding MDD of indapamide is 2.5 mg hence AI limit would be 12.5 ng/day for the “2-methyl-1-nitrosoindoline”.
If we apply CPCA approach for the “2-methyl-1-nitrosoindoline” then it also falls under category-4 and AI limit would be 1500 ng/day.
what i believe is that the toxicity of “2-methyl-1-nitrosoindoline” is not due to the nitroso structure but might be due to some other functionality or re-arrangement as described in Ponting and Foster .
so my question is, all the three indoline structures are more or less similar and “2-methyl-1-nitrosoindoline” is already having AI limit of 12.5 ng/day.
In this case,…
is it the correct way to apply CPCA approach and categorise “1-nitrosoindoline” and “3-methyl-1-nitrosoindoline” as category-4 with 1500 ng/day as AI limit?
or
should we stick to default AI limit of 18 ng/day for the “1-nitrosoindoline” and “3-methyl-1-nitrosoindoline” ?
or
should we consider AI limits 12.5 ng/day for the “1-nitroso indoline” and “3-methyl-1-nitrosoindoline” as per “2-methyl-1-nitrosoindoline”?
Unfortunately I don’t have access to the EP monographs to read their rationale, but my understanding is that the limits in them are specification limits taking a number of factors into account, not strictly the regulator-requested AI limits. This compound lacks compound-specific carcinogenicity data to derive a limit from, so it must have been found in some other way, and I would definitely hope that EP take the CPCA into account when they next update the monograph.
My best guess would be that there are multiple nitrosamine impurities, which results in different treatment of the limits (EMA Q&A section 10) in the specification for each, but the AI limits for the compounds in the abstract case are not changed.
As a result, I would say that back-conversion from a DP-specific monograph for general AI limit determination for any compound is not an appropriate, especially as it is in this case implying a limit less than 18 ng/day which was the limit before the CPCA was developed, and argue even more strongly against taking a read-across from this value to other compounds where the default CPCA is 1500 ng/day.
Should a Fischer-Hepp rearrangement occur, as I described in Ponting & Foster, the potency of the resultant C-nitroso compounds is not cohort of concern-worthy - so again the limit if mutagenic would be 1500.
As a result I would argue strongly that the AI limit for all three indoline compounds should be 1500 ng/day in the general case. (as an aside, 2-methyl-1-nitrosindoline is actually category 5, lacking an a-CH2 group)
I’d like to read the justification from the monographs to see where that exceptionally low specification limit came from, if anyone can DM the relevant section to me!
