AIs for Complex Nitrosamines by MW Adjustments, any experience?

With the recent publication of FDA’s Updates on possible mitigation strategies to reduce the risk of nitrosamine drug substance-related impurities in drug products | FDA
A new concept have emerged: ‘nitrosamine drug substance-related impurities (NDSRIs)’ a class of nitrosamines sharing structural similarity to the API

For this class of nitrosamines, the read-across when setting limits can be quite challenging. We have seen the case of N-nitroso-varenicline (37 ng/day). This afternoon Dr. Nudelman presented the use of MW adjustment and ratios as a potential path to set AIs for complex Nitrosamines. @conudel would you help us understand bit more about that concept and how has been the regulatory acceptance of this concept? Thx

For our community members, any experience to share on the use of this option?

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Worth noting: This concept (MW adjustment) has been supported as scientifically appropriate for TTC because the chemical activity should be considered on a molar (vs MW) basis. It has not (as yet) been taken up by regulators mostly because the initial analyses to support TTC were based on MW and regulators are used to setting limits based on MW. But, the principal is acknowledged as being scientifically supported (and in fact, preferred). This is described in the 2015 WHO/EFSA workshop: []


I think it has also been acknowledged in some guidance documents for read across. For example, from an ECETOC doc on Read Across (, it is mentioned that it should be done on a molar basis:


@SusanFelter already provided some important publications on the topic.
Furthermore, in the Memorandum of Meeting from the FDA & Joint Industry Meeting that took place on May 4, 2021, industry proposed that FDA consider molecular weight considerations by applying a default AI (molar AI) for compounds with a related structure. FDA also answered to a question asking “When using read across to derive an AI for a nitrosamine with insufficient carcinogenicity data, what specific scientific considerations does FDA believe are most important?” by stating that the overall molecular weight should be considered one of the factors in using read-across approach to derive an AI for structurally complex nitrosamines or nitrosamines without safety data (
Additionally, Aisar Atrakchi from FDA presented in the PhRMA workshop in September 2021 described the overall molecular weight and physicochemical parameters as of of the factors that should be taken into account when selecting appropriate reference compounds for read-across analysis (


In addition, scientifically it makes sense to use a molar conversion because after all we are talking about the number of nitrosamine molecules that a person is exposed to and not their weight. The bioburden causing mutations (without taking into account any other considerations) is a function of the number of nitrosamines that convert to diazonium that eventually react with DNA. A complex nitrosamine with a higher molecular weight has the same number of nitrosamine functional groups as a small nitrosamine and it will give the same overall number of mutations. That is the ultimate measure and not the weight of the nitrosamine. If anything, the higher molecular weight can only interfere with the CYP mediated bioactivation and also sterically hinder the reaction with with DNA. So overall the molecular weight has a large effect on the mutagenic activity.