The purpose of an In Vitro Mammalian Cell Mutation Assay for Nitrosamines is to evaluate the mutagenic potential and genotoxicity (structural aberrations) of nitrosamines using a mammalian cell system. This is required to fulfill regulatory requirements per the US FDA’s issued guidance on acceptable intake limits for nitrosamine drug substance related impurities (NDSRIs) in order to ensure drug safety. While there are acceptable intake (AI) limits for many nitrosamines available that are based on predicted carcinogenic potency and data analysis, in vitro mutagenicity data is not always available for test-article-specific nitroso compounds. To fulfill this requirement, multiple streams of data must be provided.  These data are typically comprised of an enhanced Ames test, a second in vitro mammalian cell mutation assay for nitrosamines, and in vitro metabolism data (in human hepatocytes or microsomes).

How the In Vitro Mammalian Cell Mutation Assay for Nitrosamines is Performed

For the required in vitro mammalian cell mutation assay for nitrosamine drug substance related impurities, also referred to as the Mouse Lymphoma assay for NDSRIs, Nucro-Technics recommends performing a derivative of the OECD 490 guideline, “In Vitro Mamallian Cell Gene Mutation Tests Using the Thymidine Kinase Gene”. While similar to a traditional OECD 490 test, it includes the use of additional nitrosamine controls with metabolic activation (S9). The use of these additional controls is critical as standard assay conditions may sometimes show reduced sensitivity for certain nitrosamines due to insufficient activation of CYP enzymes in S9 condition. These controls can also be used to support a weight-of-evidence approach that would be required for any type of nitrosamine risk assessment.

The in vitro mammalian cell mutation assay for nitrosamine, performed in L5178Y mouse lymphoma cells, is a key in vitro mammalian cell mutation test that detects and confirms the mutagenic potential induced by chemical substances as well as their metabolites. A positive result for this assay would indicate that the nitrosamine in question induces gene mutations at the TK gene locus in the cultured mammalian cells used.

Nucro-Technics is well equipped to complete these studies. Our organization has been performing OECD 490 tests for decades. We have also been performing nitrosamine impurities testing since early 2019, when regulatory agencies, like the FDA, began asking for this type of analysis. Variations of these studies have been performed for dozens of companies and for hundreds of APIs and marketed products. This test is conducted in compliance with Good Laboratory Practice regulations and can be used to support regulatory submission filings.