Optimizing the detection of N-nitrosamine mutagenicity in the Ames test -Pub

New Scientific Knowledge & Development
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Dr. Heflich et.al from FDA published the literature on optimizing the Ames test.

Highlights

  • 12 small molecule nitrosamines and 17 NDSRIs were assessed for mutagenicity in the Ames test

  • Different test protocols were evaluated for their effect on assay sensitivity.

  • A combination of TA1535 and WP2 uvrA(pKM101) was able to detect all mutagenic nitrosamines

  • In general, preincubations with 30% S9 produced higher mutagenic responses than 10% S9

  • In general, hamster liver S9 produced higher mutagenic responses than rat liver S9

  • Factors affecting assay sensitivity were similar for NDSRIs and small molecule nitrosamines

These test variables were evaluated by testing 12 small-molecule N-nitrosamines and 17 NDSRIs for mutagenicity in Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537, and Escherichia coli strain WP2 uvrA (pKM101). Eighteen of the 29 N-nitrosamine test substances tested positive under one or more of the testing conditions and all 18 positives could be detected by using tester strains TA1535 and WP2 uvrA (pKM101), preincubations of 30 minutes, and S9 mixes containing 30% hamster liver S9.

Overall responses

Ten of 12 small molecule nitrosamines (83%) and eight of 16 ‘testable’ NDSRIs (50%) were positive in the Ames test under one or more of the 50 combinations of tester strain, activation condition, and preincubation times that were used. One NDSRI (N-nitroso-ciprofloxacin) was difficult to test because of its bacterial toxicity and is not

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Yosuke, thanks a lot for your input.
The percentage of positive Ames tests (50%) for the NDSRIs is high, proving that Ames test is not the best solution to extend the AIs of them, as it was generally beleived sometime ago.
As, for the moment, i cannot have access to this paper, do you know if any of the Ames test which are decribed in, is identical to ehnanced Ames test?
thanks in advance
Christos

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The authors stress that the high prevalence of Ames positives in the data set is to be interpreted with care, as no effort for randomization of choice of nitrosamines was made.

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Does anyone know if this was the data/testing used for part of the poster that the FDA presented earlier this year?
It looks like it is the same NDSRIs that are producing negative results as the poster.
That poster included an additional, more advanced test methodology that the AMES test as well - is there a second paper on the way?

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thanks a lot for your clarification Wybon, it is much appreciated.

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I want to add a rough summary of 12 small nitrosamines and 17 NDSRIs. The original should be checked for details.

Small nitrosamines with Posi(10): NDMA, NDEA, NIPEA/NEIPA, MNP, NDIPA, NMPA, NEPA, NDBA, CPNP, N,N-bis(2,2-diethoxyethyl) nitrous amide

Small nitrosamines with Nega(2): NMBA, N-nitroso-diphenylamine

NDSRIs with Posi(8): N-nitroso-lorcaserin, N-nitroso-varenicline, N-nitroso-desmethyl diphenhydramine, N-nitroso-propranolol, N-nitroso-nortriptyline, N-nitroso-duloxetine, N-nitroso-sertraline, N-nitroso-fluoxetine,

NDSRIs with Nega(8): N-nitroso-phenylephrine, N-nitroso-diclofenac, N-nitroso-paroxetine, N-nitroso-desvaleryl valsartan, N-nitroso-bumetanide, nitroso-desvaleryl valsartan methyl ester, N-nitroso-folic acid, N-nitroso-dabigatran etexilate

NDSRI not tested: N-nitroso-ciprofloxacin (highly toxic to tester strains, poorly soluble in all vehicles)

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I think it is.
I interpret March 2024 SOT P194 as a preview of the data (but focusing more on the in vitro follow ups), the Xilin Li May 2024 presentation contained the extended examples and contained also some shared graphics with the paper (Figure 1 and 2 of Heflich 2024). Li 2023 paper (N-nitroso-propranolol) was the start of all this and N-nitroso-propranolol is not added in the supplementary information of Heflich 2024 (reference to Li 2023), though Heflich 2024 paper clarifies some more experimental details on the set-up for the Li 2023 Ames test I believe. From Li 2023 to Heflich 2024, I believe the experimental set-up is consistent (also with organised workshop), only hiccup in the road seemed to be availability problems with Aroclor 1254 induced S9 and a full shift to PB/NF induction (but with careful confidence in the replaceability of Aroclor 1254 and PB/NF induction; in 3% of the cases a greater response with Aroclor 1254 induction).

The full set of in vitro follow-ups of March 2024 SOT P194 (like Li 2023 on N-nitroso-propranolol) might get their own paper still indeed, but there are also some actions for evaluating of further modification of the Ames test which have been identified for possible future work. Noteworthy, the N-nitroso-propanolol in vitro follow-ups are again cited, including the CYP-specific evaluations with genome edited TK 6 cells, confirming CYP-mutagenicity positive evaluation is indirect proof only for alpha-hydroxylation: recognition that it cannot be proven that the indirect acting mutagenicity is nitroso-related (but some remarks on matching strain specificity and activation trends with nitrosamines) (“nitrosamine-typical”). I believe that realization that the expensive testing with CYP-expressing TK 6 cells doesn’t proof nitroso-related mutagenicity directly, can be connected with the suggestion to also look into modification of the Ames test with CYP-specific expressing bacteria strains or chemical activation systems (cheaper but can also be CYP-supporting?). Overall, the realization communicated in this paper that in vitro positives do not always have an in vivo relevance or might not always be nitroso-related is important to advance further design of testing and mechanistic interpretation.

Overall an excellent choice to focus on the Ames test alone, this allowed maximizing the sharing of data on Ames test I believe.

Noteworthy, while the authors discuss it remains uncertain if an Ames test positive is truly indicative for a cancer risk (allowing linking with in vivo (or in vitro) follow-up data), the reverse scenario, the value of a negative Ames test is not discussed in this work and could get more attention again when papers on follow-up testing appear. Positives and the protocol are really the center of attention here.

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This was a special case linked to solubility/bioavailability constraints and “nitrosamine-typical” results and should indeed be checked in detail in the manuscript.

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Dear Wybon,

Thank you for the detailed information. It helps me to understand this paper deeply. :grinning: :grinning: :grinning:

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I would like to highlight also that N-nitrososertraline was found positive in this Ames testing while before some months ago (on July), in the EMA appendix 1, its AI has been set as 1500ng/day due to negative bacterial reverse mutation test.
Of course, in the last update, its limit was decreased to 100ng/day as the substance tested positive in in vivo mutagenicity study.
thank you

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Chris, it was good that you raised the nitroso sertraline issue. I have clients who originally were controlling it at 1500 ng/day and now have to tighten the limits to 100 ng/day and are struggling. I agree that trying to find short cuts like modified AMES (as my grand advisor Dr. Lijinsky always said, Ames is not the best for nitrosamines), I wish they plunged into in vivo studies of representative NDSRIs (may be 20-25) and then use read across. If this started in 2020, we would have had a solution by now.

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Yusukemino San, you are an angel. thanks for this summary. It is incredibly helpful.

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This also explains the invite only conference FDA and HESI are having at White Oaks on Oct 15-16. Usually such conferences are recorded, so we will possibly see the transcript. If anybody in this group is invited, please provide us a high level summary of what was discussed.

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thanks Aloka,
in my opinion your suggestion for setting AIs through a read-across approach using in-vivo tested NDRSIs, is sounds to me more reasonable.
Christos

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Thank you, @chrischar and @ASrinivasan.
I guess EAT and other safety tests will be discussed in the nitrosamine workshop co-hosted by FDA and CRGC.

Session 1: Safety & Risk Assessment Methods and Recommendations for Acceptable Intake Limit

This session will discuss the current research regarding safety testing methods for NDSRIs and recommended acceptable intake limits for NDSRIs based on predicted carcinogenic potency and compound-specific data or read-across analysis.

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