Strategies for Assessing Acceptable Intakes for Novel N-Nitrosamines Derived from Active Pharmaceutical Ingredients

Abstract

The detection of N-nitrosamines, derived from solvents and reagents and, on occasion, the active pharmaceutical ingredient (API) at higher than acceptable levels in drug products, has led regulators to request a detailed review for their presence in all medicinal products. In the absence of rodent carcinogenicity data for novel N-nitrosamines derived from aminecontaining APIs, a conservative class limit of 18 ng/day (based on the most carcinogenic N-nitrosamines) or the derivation of acceptable intakes (AIs) using structurally related surrogates with robust rodent carcinogenicity data is recommended. The guidance has implications for the pharmaceutical industry given the vast number of marketed amine-containing drugs. In this perspective, the rate-limiting step in N-nitrosamine carcinogenicity, involving cytochrome P450-mediated α-carbon hydroxylation to yield DNA-reactive diazonium or carbonium ion intermediates, is discussed with reference to the selection of read-across analogs to derive AIs. Risk-mitigation strategies for managing putative N-nitrosamines in the preclinical discovery setting are also presented.

Drawing a Line: Where Might the Cohort of Concern End?

“…As described in this Perspective, the Cohort of Concern as originally envisaged contained all N -nitroso compounds; however, even at the time it was noted that a substantial proportion of N -nitroso compounds were of lower potency than the TTC, and subsequent research has confirmed that the carcinogenic potencies of nitrosamines span no fewer than 4 orders of magnitude . Those N -nitroso compounds that do lead to potent carcinogenicity are those that can form a diazonium ion in close proximity to DNA and thus lead to its alkylation; their potency is related to the facility with which that diazonium can be formed. ,, As a result, the following observation can be made: not all N -nitroso compounds should be in the CoC, and indeed, not even all N -nitrosamines should be in the CoC considering that the presence of an α-hydrogen is critical for high carcinogenic potency.…”

“…The Ames test is ultimately used as a proxy for potential carcinogenicity under ICH M7, and it is worth noting that the predictivity of this assay is significantly higher , for N -nitrosamines than it is for other genotoxic carcinogens. In addition to this work by Trejo-Martin et al, ongoing efforts from the FDA, the EMA MutaMind Project, and HESI GTTC seek to confirm the optimal conditions (OECD TG471 allows for a variety of approaches) for N -nitrosamine detection in the Ames test …”

“…The critical metabolic step in the activation of N -nitrosamines is the hydroxylation of the α-carbon, − catalyzed by a variety of cytochrome P450 enzymes. Other metabolic hydroxylation reactions are possible with some nitrosamines, , but it is generally this α-hydroxylation that leads to the formation of the highly reactive alkyldiazonium cation.…”

“…2,2,6,6-Tetramethylpiperidine has been reported negative when tested in the Ames strains most sensitive to N -nitrosamines and carcinogenicity assays . However, the negative results for these and a decreased level of carcinogenic prevalence or potency ,,,,, even for hindered, but α-hydrogen-containing, nitrosamines (covered separately in section 4) combine with the mechanistic impossibility of diazonium formation to result not just in their exclusion from the CoC but also in a confident negative call for carcinogenicity (absent other toxicophores).…”

“…In addition to this work by Trejo-Martin et al, 74 ongoing efforts from the FDA, the EMA MutaMind Project, and HESI GTTC seek to confirm the optimal conditions (OECD TG471 allows for a variety of approaches 56 ) for N-nitrosamine detection in the Ames test. 22 Some of the compound classes discussed in this Perspective, however, do not require metabolic activation; 25,26 these are the nitrosoureas and primary amines. An Ames result for these compounds should not fall into the same concern with respect to the adequacy of the metabolic activation step, and the compounds should be expected, if mutagenic, to be positive in those experimental runs that do not incorporate S9 (contrary to dialkyl N-nitrosamines, which are expected to be positive only in the presence of S9).…”