R.A. Jolly et al.

Regulatory Toxicology and Pharmacology 152 (2024) 105672

Fig. 2. Cytochrome P450 catalyzed mechanism of potential DNA damage by dialkyl nitrosamines.

correlated to stretch vibrational frequencies measured by infrared (IR)
spectroscopy. Therefore, we selected C–N stretching frequencies as a QM
descriptor and attempted to find correlation with TD50. Based on both
the structural similarity of the chemicals and known metabolism of the
APIs, it was considered that alpha hydroxylation would be similar across
the NDSRIs and therefore not as impactful to the overall metabolic
activation.
The C–N bond stretch vibrational frequencies of the diazonium intermediates formed from the nitrosamines shown in Fig. 3 are evaluated.
Compounds with or without an oxygen substitution at the β and γ carbon
atoms, an EWG group at these positions, or an aromatic group were
included in this analysis (Fig. 3)
The calculated frequencies of the various diazonium intermediate
C–N bonds showed a strong correlation with their respective TD50
values: correlation coefficient = 0.89 as shown in Fig. 4. This quantum
mechanical modeling predicted a TD50 of 1.14, 1.15 and 1.19 mg/kg for
NFLX, NATX and NDLX respectively as shown in Table 2.

3.3. Bacterial reverse mutation (Ames) assay
The results of the in vitro mutation Ames assay for the NDSRIs are
shown in Table 3. All three NDSRIs were positive in strains associated
with point mutations (TA100 or TA1535) in both induced rat and
hamster S9 conditions. Consistent with other nitrosamines (Bringezu
and Simon, 2022), it was evident that metabolism was largely required
for activation and that the strains that were positive were those sensitive
to point mutation. Assay sensitivity was enhanced with the hamster (vs
rat) S9, but rat S9 also yielded a positive response for all NDSRIs. The
positive Ames data for the NDSRI compounds indicated that they have
the potential to be carcinogenic. These results confirmed the need for a
sensitive analytical method for detection of these NDSRIs in the drug
product.
3.3.1. Transgenic rat mutagenicity study
In the 7-day range finding studies, clinical observations of toxicity
and decreases in body weight, body weight gain, and food consumption
demonstrated that 100 mg/kg dose was the maximal tolerated dose

Fig. 3. Parent compounds, putative diazonium intermediates, and infrared (IR) stretching frequencies of the C–N bond of the diazonium intermediates for close
analogs of fluoxetine NDSRI based on structural and electronic similarity.
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