Health Canada: Nitrosamine impurities Update July 26, 2024
An updated version of Health Canada’s list of established Acceptable Intake (AI) limits (Appendix 1) has been posted online. The updates to Appendix 1 include:
nine (9) additional nitrosamine impurities and their corresponding CPCA-derived AI limits,
revised AI limits for three (3) nitrosamine impurities (N-nitroso-ciprofloxacin, N-nitroso-*N-*desmethyl-citalopram, and *N-*nitroso-sertraline (of note, the AI limit for N-nitroso-sertraline is reduced from 1,500 ng/day to 100 ng/day)), and
a line listing for entacapone drug substance to indicate that NDEA (AI limit = 26.5 ng/day) is a structurally-related impurity.
The decrease in the limit for N-nitroso-sertraline is intriguing; this was based on negative mutagenicity data - does this indicate that HC have received positive data for this compound…? I would be intrigued to see the details of this study if so!
23 October 2023: 1500 ng/day based on EAT (following earlier acceptance of EAT by TGA and EMA)
26 July 2024: 100 ng/day based on CPCA category 2
It is unfortunate that no short motivation is listed (to allow assessment of future impact on other cases) or transitional limits are instated.
An EAT positive/equivocal motivation with no experimental root cause for the outcome would be very strange indeed.
I find it noteworthy that the EAT negative on N-nitroso-dorzolamide (structural resemblance and shared CPCA category) remains intact, possibly making the data to build the decision on very specific.
Overall the July publications from HC, TGA and EMA still give confidence in the endorsement of the EAT (the later TGA update leaves the EAT on nitroso-sertraline intact). (So far, EMA/HC/TGA congruence on limits was around 80%).
Could this be a reflection of in vivo taking priority over in vitro but regulators not being ready yet to deal with proposed AI derivatisation based on positive in vivo mutagenicity data?
(Though it seems to me exceptional to not be able to support AIs above 1500 ng/day, making a temporary go-back to 100 ng/day probably unnecessary burdensome, especially considering that also the CPCA explanatory paper of NITWG shows that the alpha-benzylic feature is overvalued. Maybe other regulators feel more comfortable with 1500 ng/day in case of an in vivo positive or equivocal EAT that is being investigated).
Or mutagenicity warnings from further in vitro studies (which can be overalarming as seen by the fluoxetine case)?
It is still early to see and explain a definite pattern, but I couldn’t help but notice that the current volatility in potency assignment (in literature and by regulators; by looking at new in vitro and in vivo data) seems to be bigger for NDSRIs for which the API itself is known to inhibit CYP enzymes with specifically an at least weak inhibitory effect on CYP2C19, so far one of the most commonly identified bioactivators for NDSRIs based on in vitro studies.
(Sertraline is known to inhibit several CYP enzymes, including a weak inhibitory effect on CYP2C19. This property is shared with fluoxetine for example. Dorzolamide is not known for its inhibitory effect on CYP2C19 I believe.)
Another property that NDSRIs with volatility in potency assignment often have, is the presence of aromatic rings. Aromatic hydroxylated metabolites can sometimes form reversible adducts with macromolecules, which limits the ultimate biological impact in vivo, but forms a possible explanation for in vitro mutagenicity should this be the case.
Thank you for the updates. The case of entacapone contaminated with NDEA may reflect recalls in Japan.
We need clarification about the AI of N-nitroso-sertraline. If positive in vivo test results replace negative EAT results, the advantage of conducting in vivo tests will be lost. If so, I hope the authorities allow us to establish an acceptable intake of nitrosamines through positive in vivo tests.
We need to keep a close eye on further information about the AI of N-nitroso-sertraline.
dear team,
i think the case of entacapone is reasonable as diethylamine it could be a degradation product (due to hydrolysis of the amidic bond).
Regarding Sertraline, i think that Wybon describes the situation very accurate.
Do you think that there is a possibility the EAT test which has been used to rise the limit to 1500ng/day, finally to be proved unreliable and not be accepted in a second round of assessing?
thank you
If someone followed up with an In-vivo and the results are positive then is plausible to see the limit going back to 100 ng/day, as for the time being +tive in-vivo to define limits is an on-going discussion.
Diego thanks for your feedback.
i think that Wybon describes this possibility also before, and i agree that it is the most plausible one.
I just wondering if it could be a QA issue with the Ames test.
best regards
thanx a lot Estefanny,
just note that the updated of the AI of N-Nitroso-desmethyl-citalopram from 18 to 100ng/day is out of explanation (most probably from read-across approach?)
best regards
Christos
best regards
The harmonisation degree between the EMA and TGA limit list is at the moment 98%, it seems the TGA uses the NITWG forum effectively to strive for harmonisation with the EMA. With HC, the harmonisation degree is 85%.
The TGA limit list is mostly based on adoption of EMA RAILs (in so far relevant for this territory) and on their website TGA also indicates endorsing EMA guidance. EMA first and then TGA is what we typically see, unless maybe if HC is sooner and TGA follows already (exceptional).
In that context, it can indeed be evaluated that in July 2024 the TGA followed the EMA reporting in the same month to go from CPCA category 1 to NNK readacross for N-nitroso-desmethyl-citalopram. I would thus read the EMA footnote “Limit derived using structure-activity-relationship (SAR)/read-across approach using the TD50 of NNK as point of departure.” with the TGA publication.
Now also realising that possibly the HC RAIL change on N-nitroso-sertraline is linked to the FDA Ames test results reported in May 2024: N-nitroso-sertraline is uniquely positive in E. coli WP2 uvrA (pKM101), only with the use of hamster S9 (10% and 30%) in these tests.
Fiscal Year (FY) 2024 Generic Drug Science and Research Initiatives Public Workshop. Presentation “N-Nitrosamine Drug Impurity Research at FDA/NCTR: Assessing the Mutagenicity of N-Nitrosamines and NDSRIs” - Xilin Li, PhD, Visiting Scientist, DGMT, NCTR, FDA. https://www.fda.gov/media/177881/download
Thank you for sharing the information, Wybon. The unique positive results of E. coli WP2 uvrA (pKM101), only with hamster S9 (10% and 30%) are interesting. I am not an expert on toxicity, but I thought other strains were more sensitive to nitrosamines. I add a poster presentation by Dr. Nisar.
Validation of enhanced Ames assay using N-nitrosamine positive controls
I am interested in the next updates of Appendix 1 by EMA.
For small nitrosamines, typically TA100, TA1535 and E. coli WP2uvrA (pKM101) (detecting base-pair substitutions, but importantly with E. coli WP2uvrA (pKM101) also other changes indicatable) are the most sensitive strains based on the available public data. Especially for acyclic nitrosamines, E. coli WP2uvrA (pKM101) is considered important.
For the dataset including small nitrosamines and NDSRIs, FDA scientists indicated TA1535 and E. coli WP2uvrA (pKM101) as most sensitive strains in May 2024. No nitrosamine was uniquely mutagenic in TA100, TA98 or TA1537 in these studies.
In cases where unique positivity for a certain subset of conditions was seen in E. coli WP2 uvRA (pKM101) in the state of the art on small nitrosamines, this was often linked with experimental limitations, e.g.:
When using rat liver S9 mix, E. coli WP2 uvRA (pKM101) is the most sensitive strain for NDEA. But compare Thomas 2024 to Dieckhoff 2024 conditions and results (additional + in TA100, TA1535 when increasing rat S9, lowering vehicle concentration and applying different preincubation conditions). Redirectinghttps://doi.org/10.1093/mutage/gead033Redirecting
o Possible limitation: CYP2E1 enzyme capacity in rat liver and CYP2E1 DMSO inhibition
So overall it is useful that E. coli WP2 uvRA (pKM101) is an OECD 471 included strain (older Ames studies on nitrosamines often relied on TA100 and TA1535), to maximize the overall sensitivity of the EAT and increase confidence in the Ames test negativity.
But this doesn’t mean that false positives (or conservative conclusions by going back to CPCA due to the sensitivity in E. coli WP2 uvRA (pKM101)) are not possible (especially with a strain capable to show a wider range of mutations) and a full review and comparison of experimental conditions for the studies available remains needed. The in vivo validation of the sensitivity of E. coli WP2 uvRA (pKM101) has mostly been reported for small nitrosamines and extrapolation to all NDSRIs is not fully warranted.
Sertraline itself has been described to induce mutations in bacteria, just like fluoxetine (reactive oxygen species mediated mutagenesis). While I don’t have data showing that this can be picked up in the EAT, an API co-run in E. coli WP2 uvRA (pKM101) could be useful for this class of APIs.
Biochemically the link between fluoxetine and sertraline remains noteworthy (see also previous CYP2C19 remark). It seems that for N-nitroso-fluoxetine (see also 4400 ng/day based on in vivo study and potency ranking) FDA data suggests E. coli WP2uvrA (pKM101) also to be the most sensitive strain looking at study summaries, albeit not the uniquely positive strain. In Industry studies, positivity with E. coli WP2 uvRA (pKM101) was seen in 10% rat S9, not in 10% hamster. Based on consistent positivity in TA1535 and TA100 across activations and analogues, Industry paper suggests these rather as most sensitive strains for N-nitroso-fluoxetine. https://www.sciencedirect.com/science/article/pii/S0273230024001132?via%3Dihub
Thank you for the detailed explanation, Wybon. We should consider the profiles in these conditions to avoid false positives in the EAT for NDSRIs. I understand E. coli WP2uvrA (pKM101) plays an important role in the test. The structures of NDSRIS are also important.
For N-nitroso sertraline, it was once classified as negative in EAT. False negatives also should be avoided. It is a big challenge.
