N-Nitrosamine drug substance related impurities (NDSRIs) - A proposal for the addition of subcategories to carcinogenic potency categorization approach categories 1 and 2 for NDSRIs with a molecular weight > 200 Da.

The carcinogenicity potency categorization approach (CPCA) derived and harmonized by Health Authorities was a significant milestone, as it defined molecular properties that allowed for the rapid evaluation of the chemical structures of N-nitrosamine drug substance related impurities (NDSRIs) and the assignment of associated lifetime Acceptable Intake (AI) limits to inform on appropriate impurity control strategies in certain drug products. Nonetheless, it is important to continue to refine and improve on the CPCA based upon data-derived evidence. Herein, we focus on the default CPCA AI for NDSRIs, which is largely based on the small molecule N-nitrosamines (NAs).

Evaluation of the nitrosamine impurities of ACE inhibitors using computational, in vitro, and in vivo methods demonstrate no genotoxic potential.

Evaluation and mitigation of the potential carcinogenic risks associated with nitrosamines in marketed pharmaceutical products are areas of interest for pharmaceutical companies and health authorities alike. Significant progress has been made to establish acceptable intake (AI) levels for N-nitrosamine drug substance-related impurities (NDSRIs) using SAR, however some compounds require experimental data to support derivation of a recommended AI. Many angiotensin-converting enzyme inhibitors, identified by the suffix "pril," have secondary amines that can potentially react to form nitrosamines.

Mechanisms of Nitrosamine Mutagenicity and Their Relationship to Rodent Carcinogenic Potency.

A thorough literature review was undertaken to understand how the pathways of -nitrosamine transformation relate to mutagenic potential and carcinogenic potency in rodents. Empirical and computational evidence indicates that a common radical intermediate is created by CYP-mediated hydrogen abstraction at the α-carbon; it is responsible for both activation, leading to the formation of DNA-reactive diazonium species, and deactivation by denitrosation. There are competing sites of CYP metabolism (e.

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

The detection of -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 -nitrosamines derived from amine-containing APIs, a conservative class limit of 18 ng/day (based on the most carcinogenic -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.

Practical and Science-Based Strategy for Establishing Acceptable Intakes for Drug Product -Nitrosamine Impurities.

The potential for -nitrosamine impurities in pharmaceutical products presents a challenge for the quality management of medicinal products. -Nitrosamines are considered cohort-of-concern compounds due to the potent carcinogenicity of many of the structurally simple chemicals within this structural class. In the past 2 years, a number of drug products containing certain active pharmaceutical ingredients have been withdrawn or recalled from the market due to the presence of carcinogenic low-molecular-weight ,-dialkylnitrosamine impurities.

Permitted daily exposure limits for noteworthy N-nitrosamines.

A genotoxic carcinogen, N-nitrosodimethylamine (NDMA), was detected as a synthesis impurity in some valsartan drugs in 2018, and other N-nitrosamines, such as N-nitrosodiethylamine (NDEA), were later detected in other sartan products. N-nitrosamines are pro-mutagens that can react with DNA following metabolism to produce DNA adducts, such as O -alkyl-guanine. The adducts can result in DNA replication miscoding errors leading to GC>AT mutations and increased risk of genomic instability and carcinogenesis.

Deriving Compound-Specific Exposure Limits for Chemicals Used in Pharmaceutical Synthesis: Challenges in Expert Decision-Making Exemplified Through a Case Study-Based Workshop.

A workshop entitled "Deriving Compound-Specific Exposure Limits for Chemicals Used in Pharmaceutical Synthesis" was held at the 2018 Genetic Toxicology Association annual meeting. The objectives of the workshop were to provide an educational forum and use case studies and live multiple-choice polling to establish the degree of similarity/diversity in approach/opinion of the industry experts and other delegates present for some of the more challenging decision points that need to be considered when developing a compound-specific exposure limit (ie, acceptable intake or permissible or permitted daily exposure). Herein we summarize the relevant background and case study information for each decision point topic presented as well as highlight significant polling responses and discussion points.

Management of pharmaceutical ICH M7 (Q)SAR predictions - The impact of model updates.

Pharmaceutical applicants conduct (Q)SAR assessments on identified and theoretical impurities to predict their mutagenic potential. Two complementary models-one rule-based and one statistical-based-are used, followed by expert review. (Q)SAR models are continuously updated to improve predictions, with new versions typically released on a yearly basis.

In Vivo Mutagenicity Testing of Arylboronic Acids and Esters.

Arylboronic acids and esters (referred to collectively as arylboronic compounds) are commonly used intermediates in the synthesis of pharmaceuticals but pose a challenge for chemical syntheses because they are often positive for bacterial mutagenicity in vitro. As such, arylboronic compounds are then typically controlled to levels that are acceptable for mutagenic impurities, that is, the threshold of toxicological concern (TTC). This study used ICH M7 guidance to design and conduct a testing strategy to investigate the in vivo relevance of the in vitro positive findings of arylboronic compounds.

Genetic toxicology in silico protocol.

In silico toxicology (IST) approaches to rapidly assess chemical hazard, and usage of such methods is increasing in all applications but especially for regulatory submissions, such as for assessing chemicals under REACH as well as the ICH M7 guideline for drug impurities. There are a number of obstacles to performing an IST assessment, including uncertainty in how such an assessment and associated expert review should be performed or what is fit for purpose, as well as a lack of confidence that the results will be accepted by colleagues, collaborators and regulatory authorities. To address this, a project to develop a series of IST protocols for different hazard endpoints has been initiated and this paper describes the genetic toxicity in silico (GIST) protocol.

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.

Principles and procedures for handling out-of-domain and indeterminate results as part of ICH M7 recommended (Q)SAR analyses.

The International Council for Harmonization (ICH) M7 guideline describes a hazard assessment process for impurities that have the potential to be present in a drug substance or drug product. In the absence of adequate experimental bacterial mutagenicity data, (Q)SAR analysis may be used as a test to predict impurities' DNA reactive (mutagenic) potential. However, in certain situations, (Q)SAR software is unable to generate a positive or negative prediction either because of conflicting information or because the impurity is outside the applicability domain of the model.

In silico toxicology protocols.

The present publication surveys several applications of in silico (i.e., computational) toxicology approaches across different industries and institutions.

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).

Potential impurities in drug substances: Compound-specific toxicology limits for 20 synthetic reagents and by-products, and a class-specific toxicology limit for alkyl bromides.

This paper provides compound-specific toxicology limits for 20 widely used synthetic reagents and common by-products that are potential impurities in drug substances. In addition, a 15 μg/day class-specific limit was developed for monofunctional alkyl bromides, aligning this with the class-specific limit previously defined for monofunctional alkyl chlorides. Both the compound- and class-specific toxicology limits assume a lifetime chronic exposure for the general population (including sensitive subpopulations) by all routes of exposure for pharmaceuticals.

Resolution of contradiction between in silico predictions and Ames test results for four pharmaceutically relevant impurities.

The ICH M7 Guideline requires low level control of mutagenic impurities in pharmaceutical products to minimize cancer risk in patients (ICHM7, 2014). Bacterial mutagenicity (Ames) data is generally used to determine mutagenic and possible carcinogenic potential of compounds. Recently, a publication on experiences of using two in silico systems to identify potentially mutagenic impurities highlighted the importance of performing a critical review of published Ames data utilized as part of a mutagenicity assessment of impurities (Greene et al.

Principles and procedures for implementation of ICH M7 recommended (Q)SAR analyses.

The ICH M7 guideline describes a consistent approach to identify, categorize, and control DNA reactive, mutagenic, impurities in pharmaceutical products to limit the potential carcinogenic risk related to such impurities. This paper outlines a series of principles and procedures to consider when generating (Q)SAR assessments aligned with the ICH M7 guideline to be included in a regulatory submission. In the absence of adequate experimental data, the results from two complementary (Q)SAR methodologies may be combined to support an initial hazard classification.

Establishing best practise in the application of expert review of mutagenicity under ICH M7.

The ICH M7 guidelines for the assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals allows for the consideration of in silico predictions in place of in vitro studies. This represents a significant advance in the acceptance of (Q)SAR models and has resulted from positive interactions between modellers, regulatory agencies and industry with a shared purpose of developing effective processes to minimise risk. This paper discusses key scientific principles that should be applied when evaluating in silico predictions with a focus on accuracy and scientific rigour that will support a consistent and practical route to regulatory submission.

A practical application of two in silico systems for identification of potentially mutagenic impurities.

The International Conference on Harmonization (ICH) M7 guidance for the assessment and control of DNA reactive impurities in pharmaceutical products includes the use of in silico prediction systems as part of the hazard identification and risk assessment strategy. This is the first internationally agreed guidance document to include the use of these types of approaches. The guideline requires the use of two complementary approaches, an expert rule-based method and a statistical algorithm.

Compensatory erythropoiesis has no impact on the outcome of the in vivo Pig-a mutation assay in rats following treatment with the haemolytic agent 2-butoxyethanol.

The Pig-a assay has rapidly gained international interest as a useful tool for assessing the mutagenic potential of compounds in vivo. Although a large number of compounds, including both mutagens and non-mutagens, have been tested in the rat Pig-a assay in haematopoietic cells, there is limited understanding of how perturbations in haematopoiesis affect assay performance. Of particular concern is the possibility that regenerative haematopoiesis alone, without exposure to a genotoxic agent, could result in elevated Pig-a mutant cell frequencies.

Evaluation of the in vivo mutagenicity of isopropyl methanesulfonate in acute and 28-day studies.

Understanding the mutagenic dose response could prove beneficial in the management of pharmaceutically relevant impurities. For most alkyl ester impurities, such as isopropyl methanesulfonate (IPMS), little in vivo mutagenicity data exist for dose analysis. The likelihood of a sublinear dose response for IPMS was assessed by comparing the Swain Scott constant, the SN 1/SN 2 reaction mechanism and the O(6) :N(7) guanine adduct ratio to that of more well-known alkyl esters.

Evaluation of the Pig-a, micronucleus, and comet assay endpoints in a 28-day study with ethyl methanesulfonate.

Ethyl methanesulfonate (EMS) was evaluated as part of the validation effort for the rat Pig-a mutation assay and compared with other well-established in vivo genotoxicity endpoints. Male Sprague-Dawley (SD) rats were given a daily dose of 0, 6.25, 12.

Impact of cell cycle delay on micronucleus frequency in TK6 cells.

Previous studies with TK6 cells have shown that extending the recovery period after pulse treatment allows for greater micronucleus expression for some compounds. This study explores the role of cell cycle delay in micronucleus expression after pulse treatment with three model genotoxins [mitomycin C, etoposide (ETOP), vinblastine]. Cells were treated for 4 hr and allowed to recover for 36 hr with samples removed at various time points during the recovery period and analyzed for cell cycle distribution, apoptosis and micronucleus frequency.