Setting limits for N-nitrosamines in drugs: A defined approach based on read-across and structure-activity relationship for N-nitrosopiperazine impurities.

This paper describes DARAN (Defined Approach for Risk Assessment of New Nitrosamines), an new defined approach that uses lines of reasoning based on structure-activity relationship (SAR) patterns and Read-Across (RAx) to set transparent and acceptable limits for new N-nitrosamines for which no toxicological data exist. We selected the compound 1-methyl-4-nitrosopiperazine (MeNP) as a target to calculate a new acceptable limit on the basis of a more transparent and scientifically reasoned RAx. We used publicly available databases and datasets to retrieve experimental in vitro mutagenicity and in vivo carcinogenicity data for N-nitrosopiperazine compounds and to form the chemical category for an RAx. We carried out SAR analyses to try to understand patterns and to obtain interpretable inferences of variation in carcinogenic potency among the N-nitrosopiperazines compounds and their differences with the potent nitrosamines NDMA (N-nitrosodimethylamine) and NDEA (N-nitrosodiethylamine). To estimate an acceptable limit for the target MeNP, we used the scientifically based hypotheses and the evidence lines of about the influence of structural attributes for a robust RAx. On the basis of the criteria proposed in the Assessment Report EMA/369136/20202 and by using the SAR hypotheses obtained by the analysis, we obtained a robust RAx, scientifically supported assumptions, which resulted in TD50 values predicted from the closest structurally related compounds and a worst-case approach.

A consortium-driven framework to guide the implementation of ICH M7 Option 4 control strategies.

The ICH M7 Option 4 control of (potentially) mutagenic impurities is based on the use of scientific principles in lieu of routine analytical testing. This approach can reduce the burden of analytical testing without compromising patient safety, provided a scientifically rigorous approach is taken which is backed up by sufficient theoretical and/or analytical data. This paper introduces a consortium-led initiative and offers a proposal on the supporting evidence that could be presented in regulatory submissions.

2-Hydroxypyridine N-Oxide is not genotoxic in vivo.

2-Hydroxypyridine N-oxide (HOPO) is an important coupling reagent used in pharmaceutical synthesis. Our laboratory previously reported HOPO as equivocal in the Ames assay following extensive testing of multiple lots of material. Given the lack of reproducibility between lots of material and the weak increase in revertants observed, it was concluded that it would be highly unlikely that HOPO would pose a mutagenic risk in vivo. The purpose of the current investigation was to assess experimentally in rats the mutagenic (Pig-a mutation induction) and more broadly genotoxic (micronucleus and comet induction) potential of HOPO. Rats were administered HOPO (0, 50, 150, 300, and 500 mg/kg/day) by oral gavage for 28 days. At the end of study, the following parameters were assessed: frequency of Pig-a mutant red blood cells and reticulocytes, frequency of peripheral blood micronuclei, and the incidence of comet formation in liver. Toxicokinetic data collected on study Days 1 and 28 demonstrated systemic exposure to HOPO. Although there were no overt clinical signs, animals treated with HOPO showed a dose-related decrease in body weight gain. There were no increases observed in any of the genotoxicity endpoints assessed. The results from this study further support the conclusion that in the context of pharmaceutical synthesis, HOPO should not be considered a mutagenic impurity but rather controlled as a normal process-related impurity. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.

2-Hydroxypyridine-N-oxide (HOPO): Equivocal in the ames assay.

2-Hydroxypyridine-N-oxide (HOPO) is a useful coupling reagent for synthesis of active pharmaceutical ingredients. It has been reported to be weakly mutagenic in the Ames assay (Ding W et al. []: J Chromatogr A 1386:47-52). According to the ICH M7 guidance (2014) regarding control of mutagenic impurities to limit potential carcinogenic risk, mutagens require control in drug substances such that exposure not exceeds the threshold of toxicological concern. Given the weak response observed in the Ames assay and the lack of any obvious structural features that could confer DNA reactivity we were interested to determine if the results were reproducible and investigate the role of potentially confounding experimental parameters. Specifically, Ames tests were conducted to assess the influence of compound purity, solvent choice, dose spacing, toxicity, type of S9 (aroclor vs phenobarbital/ß-napthoflavone), and lot variability on the frequency of HOPO induced revertant colonies. Initial extensive testing using one lot of HOPO produced no evidence of mutagenic potential in the Ames assays. Subsequent studies with four additional lots produced conflicting results, with an ∼2.0-fold increase in revertant colonies observed. Given the rigor of the current investigation, lack of reproducibility between lots, and the weak increase in revertants, it is concluded that HOPO is equivocal in the bacterial reverse mutation assay. It is highly unlikely that HOPO poses a mutagenic risk in vivo; therefore, when it is used as a reagent in pharmaceutical synthesis, it should not be regarded as a mutagenic impurity, but rather a normal process related impurity. Environ. Mol. Mutagen. 59:312-321, 2018. © 2018 Wiley Periodicals, Inc.

Trace level liquid chromatography tandem mass spectrometry quantification of the mutagenic impurity 2-hydroxypyridine N-oxide as its dansyl derivative.

A derivatization LC-MS/MS method was developed and qualified for the trace level quantification of 2-hydroxypyridine N-oxide (HOPO). HOPO is a coupling reagent used in the syntheses of active pharmaceutical ingredients (APIs) to form amide bonds. HOPO was recently confirmed to generate a positive response in a GLP Ames bacterial-reverse-mutation test, classifying it as a mutagenic impurity and as such requiring its control in APIs to the threshold of toxicological concern (TTC). The derivatization reagent 5-dimethylamino-1-naphthalenesulfonyl chloride (dansyl chloride) was used in a basic solution to convert HOPO into the corresponding dansyl-derivative. The derivative was separated from different APIs and reagents by liquid chromatography. The detection of the HOPO dansyl-derivative was achieved by mass spectrometry in selected reaction monitoring (SRM) mode. The LC-MS/MS method had a reporting limit of 0.1ng/mL HOPO, which corresponds to 0.1ppm HOPO relative to an API at 1mg/mL, and a linearity range of 0.1-25ng/mL HOPO analyte. Recoveries of HOPO standards spiked into three different API matrices at 0.2, 1.2, and 20ppm levels were all within 90-100%. An SRM-based confirmatory methodology using the ratios of two fragment ions at three CID energies was developed to verify the identity of HOPO when present at ≥0.6ppm. This identity confirmation can be employed to prevent potential false positive detection of mutagenic impurities at trace level. It can be broadly applicable for the confirmation of analytes when the analytes generate at least two major fragments in tandem mass spectrometry experiments.