Chemical Research in Toxicology

pubs.acs.org/crt

Article

Figure 1. CYP (Cytochrome P450) activated stepwise metabolic pathway for ring, alkyl, and aryl nitrosamines. Carcinogenic metabolic pathway
intermediates are labeled as A−G.

the reactivity of nitrosamines are critical to better estimate AIs
for NDSRI compounds.
Experimental data (e.g., rodent carcinogenicity) are not
available to support the derivation of a compound-specific AI
for most NDSRIs and the defaults of 18 or 26.5 ng/day are
highly conservative given that not all N-nitrosamines are highly
potent carcinogens or even carcinogenic.13 These default limits
for N-nitrosamines recommended by agencies are largely based
on the TD50 of LMW alkyl N-nitrosamines. However, the use
of read-through is dependent on the original method of AI
derivation. The derivation of the AIs that drive the default
values was developed early and many were not derived in a
consistent or transparent manner. Bercu et al.14 showed this
recently with a rederivation of AI for several N-nitrosamines.
By extension, this conservatism in AI derivation magnifies the
impact of the analogous AI read across for NDSRIs. Other
examples of conservatism in AI derivation include N-methyl-Nnitrosophenethylamine (NMPEA) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)15 where derived AIs
were calculated from pooling organ tumor data while an
approach consistent with ICH M7 would result in higher AIs.
Perhaps more important is the fact that most NDSRIs are
dramatically different from LMW alkyl N-nitrosamines based
on chemical structure.16 Differences in physicochemical
properties (such as Log P, solubility, polar surface area, etc.),
steric hindrance, expected alternate metabolism and clearance
pathways, and molecular weight are all expected to
dramatically reduce the potential for mutagenic carcinogenicity
of NDSRIs.17,18 In aggregate, these considerations argue that
AI derivation has been overly conservative for NDSRIs and

that a mechanistic analysis is one area that can be leveraged to
improve upon this.
Dobo et al.18 proposed a framework for the AI limits based
on the structure of the nitrosamine. From the common
structural features and existing nitrosamine carcinogenic data,
they divided nitrosamines into 13 groups and proposed the AI
limits conservatively in the range of 17−440 ng/day. Thomas
et al.19 developed three categories of nitrosamine potency
nitrosamine potency viz. high potency (TD50 < 0.15 mg/kg/
day), medium potency (TD50 in 0.15−1.5 mg/kg/day), and
low potency TD50 > 1.5 mg/kg/day). Recently Cross and
Ponting17 reported that the high-potency of nitrosamines is
mainly due to α-carbon hydroxylation with those nitrosamines
bearing an α-sp3-hybridized carbon. Steric and electronic
effects play a dominant role in the carcinogenic metabolic
pathway, and the interplay of these effects can result in a range
of potency, from high to low, or even a lack of carcinogenic
action. They also reported that electronic withdrawing groups
at the α-carbon significantly decrease the potency significantly.
Wenzel et al.20 recently studied the mechanism of aliphatic and
cyclic nitrosamines. They showed that hydrolysis and DNA
alkylation are competitive, and as the chain length increases the
activation energies for the hydroxylation decreases, but DNA
alkylation is more favorable thermodynamically. The work of
Wenzel et al.20 gave an understanding of how the structural
features affect toxification and detoxification reactions in the
nitrosamine metabolic pathway, while the substitution of
electron-donating, electron-withdrawing, and presence of
heteroatom requires a more detailed understanding.
Nitrosamines require metabolic activation, and the probable
carcinogenic metabolic pathway occurs through cytochrome
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https://doi.org/10.1021/acs.chemrestox.4c00087
Chem. Res. Toxicol. 2024, 37, 1011−1022