In the FDA/HESI meeting in 2023 a lot of attention was given to WoE approaches and the importance of mechanistic understanding of the metabolism (also for designing in vitro or in vivo mutagenicity studies). We are still seeing too little in vitro metabolisation assays or QM mechanistic studies in new literature or appreciation of such understanding in AI setting, would be great if the fluoxetine case would get published in literature (so that B and C get connected somehow). Yet AI setting often supposes nitrosamine selectivity or little risk for false positives (out of precaution, but if the data is there…).
N-nitroso-fluoxetine (MW relative to NNK MW is 1.633) is a great example here that in simplified test batteries you always have to try to understand what you are seeing (and in general interpretation of in vitro data including aromatic structures is often more difficult):
- Enhanced Ames test: positive (–> 30% hamster S9 and WP2 uvrA pKM 101) (FDA)
- In vitro mammalian cell gene mutation assay: positive (FDA)
- In vitro micronucleus test without S9 (with human lymphoblastoid TK6 cells): positive (FDA)
- In vitro micronucleus test with S9 (with human lymphoblastoid TK6 cells): positive (FDA)
- Human CYP activation: CYP2C19, 2B6 (FDA)
- In vitro Comet assay in primary rat hepatocytes (to try to do potency ranking with BMD approach): higher DNA damage N-nitroso-fluoxetine compared to NDMA, NDELA; N-nitroso-fluoxetine is cytotoxic (17 µM (24 h)) (Mutamind)
But then metabolic study with quantum modeling: probably identifying ether related metabolites / hydroxy-substituted or related small nitrosamines as suitable surrogate for SAR / dominant denitrosation as toxification : this might not be as potent….
And then the in vivo test (validating the quantum mechanical data?!)… it is 30 to 50 times less mutagenic than NDMA.
(The SOT 2024 poster of FDA also in general did suggest in vivo mutagenicity data as follow-up, so I don’t see why 5000 ng/day wouldn’t be justifiable if a properly conducted study is available. When doing the BMD that would be done with regards for carcinogenicity/mutagenicity correlation (and then again all the other available data (except readacross) also is focused on mutagenicity).
In vivo priority over in vitro, but in vivo should stimulate mechanistic or toxification/detoxification balance interest?
Sponsor B was definitely on to something.