NAs positive in Ames test without metabolic activation

Based on the literature, there are two types of N-nitroso compounds, the first that act directly and the second that require metabolic activation (Lijinsky, Carcinogenicity and mutagenicity of N-nitroso compounds, 1987).
Does anyone have experiences with N-nitroso compound being positive in an Ames test without metabolic activation? Is there a mechanistic explanation for this?

Thank you for answering,


@David this is for you my friend!

Great segue to David’s new paper " Strategies for Assessing Acceptable Intakes for Novel N -Nitrosamines Derived from Active Pharmaceutical Ingredients"

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 amine-containing 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.

N-nitroso compounds do indeed cover both direct and S9-requiring mutagens; the majority of the direct-acting ones are not, however, N-nitrosamines.

Nitrosoamides, nitrosoureas and others are cleaved easily in physiological conditions [Carlson 2019, @ASrinivasan and Lambert] and do not need activation.

For Nitrosamines per se, there are still a reasonable number of compounds where at least one positive result has been seen absent S9; assuming a well-conducted Ames on a pure sample, this could be expected to be where another mechanism is possible, either because another toxicophore is present, or because of some interaction of side chains with the N-nitroso functionality that modify it from the classic mechanism, as is presumably happening with NDELA. However, these cases are all positive to the best of my knowledge when studied with S9 as well.

It is definitely an indication that something beyond a simple alpha-hydroxylation is occuring, and bears further investigation - the first step of which should be a screen through an in silico tool such as Derek Nexus for other toxicophores, then some mechanistic evaluation to see if you can push electrons to yield another toxicophore or create some other intramolecular rearrangement if there are no obvious toxicophores.

Carlson 2019 (PhD thesis, U. Minnesota):
Srinavasan and Lambert: Pharmaceutical Technology - October 2022 - Pharmaceutical Technology - October 2022?