Nitrosamine Drug Substance-Related Impurities cause DNA methylation adducts in vitro and in primary hepatocytes upon Cytochrome P450-dependend metabolic activation-Pub

Limits of Nitrosamines
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Abstract

N-nitrosamines are DNA alkylating agents found in food, cosmetics, tobacco products and, more recently, drugs. Following Cytochrome P450 (CYP)-mediated metabolic activation, these compounds cause DNA damage and mutations. Unlike well-characterized compounds like N-nitrosodimethylamine (NDMA), data on the genotoxicity of nitrosamine drug substance-related impurities (NDSRIs) remain limited. Given their regulatory relevance, this study assessed the genotoxic potential of three NDSRIs —N-nitrosobetahistine (NBH), N-nitrosofluoxetine (NFluo), and N-nitrosonortriptyline (NNT) —compared to NDMA. The NDSRIs demonstrated distinct DNA methylating potential, confirmed by elevated levels of N7-methyl-deoxyguanosine (N7-MedG) and O 6-methyl-deoxyguanosine (O 6-MedG) in a DNA alkylation assay with metabolic activation. Recombinant CYP isoforms contributed differentially to the bioactivation of each NDSRI, highlighting enzyme-specific pathways of toxification. Subsequently, we demonstrated that all NDSRIs cause DNA methylation adducts (N7-MedG > O 6-MedG) in primary rat hepatocytes, with generally higher levels than those caused by NDMA. Consistently, the NDSRIs generated more DNA strand breaks than NDMA, which followed the DNA adduct kinetics. Furthermore, all NDSRIs showed cytotoxicity after 24 h, whereas no cytotoxic effect was observed for NDMA. Taken together, our study provided evidence that the three NDSRIs are genotoxic in primary rat hepatocytes, which warrants further investigation with regard to their mutagenic potential.

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To a degree I can l look at this in a positive light. N-nitroso betahistine and n-nitroso nortriptyline have both been assigned limits of 18ng/day - so lower than the limit for NDMA.

The exception to that is n-nitroso fluoxetine, which has been set a limit of 100ng/day currently.

It does back up 66% of the current scorings - is there something structural that could make the n-nitroso fluoxetine more potent as a genotoxin than the current score suggests?

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indeed is an interesting paper, but please keep in mind that there is a discrepancy between in-vitro & in-vivo results.

‘‘A mutagenicity study performed in transgenic F344 rats revealed that NFluo is mutagenic in
the liver at doses ≥30 mg/kg bw. BMD modeling resulted in a BMD of 11.4 mg/kg, whereas the BMD for liver mutagenicity was revealed to be 0.32 mg/kg for NDMA in the MutaMouse
model.

Evidently, NFluo displays higher cytotoxicity and genotoxicity than NDMA in mammalian cell models as shown in the present and in other studies, but causes lower in vivo mutagenicity in the target organ liver in rodents’’

According to this in-vivo study, the NFluo is about 35 times less potent than NDMA.

So, we should be very careful with our conclusions.

of course, is not very encouraging to see NDSRIs to present worst behaviour-even in in-vitro tests-than NDMA,

thank you,

Christos

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