Critical comparison of BMD and TD methods for the calculation of acceptable intakes for N-nitroso compounds.

The tumorigenic dose 50 (TD) is a widely used measure of carcinogenic potency which has historically been used to determine acceptable intake limits for carcinogenic compounds. Although broadly used, the TD model was not designed to account for important biological factors such as DNA repair and cell compensatory mechanisms, changes in absorption, etc., leading to the development of benchmark dose (BMD) approaches, which use more flexible dose-response models that are better able to account for these processes.

Control of N-nitrosamine impurities in drug products: Progressing the current CPCA framework and supporting the derivation of robust compound specific acceptable intakes.

The carcinogenic potency categorisation approach (CPCA) has recently been introduced by health authorities. In this model, structural features from recent literature, industry proposals, and analyses performed by health authorities, provide a rapid assessment of the potential acceptable intake (AI) for a nitrosamine impurity. As with other screening regulatory values (such as the ICH M7 Threshold of Toxicological Concern), the CPCA is conservative and can be considered a de minimis risk management framework.

Industrial Genotoxicology Group (IGG): 36th Annual Meeting Report.

The proceedings of the 36th annual meeting of the Industrial Genotoxicology Group (IGG) are shared here. The meeting held at Lhasa Limited, Leeds, UK on 28th November 2023, focussed two aspects; New Approach Methodologies (NAM's), including those for the assessment of non-standard modalities such as gas-vapour assessments and nanomaterials, and addressing the regulatory challenges associated with understanding the genotoxic and carcinogenic potential of N-nitrosamines and N-nitrosamine impurities. New approach methodologies, such as error-corrected sequencing and enhanced Ames tests that may help address these challenges were also discussed.

Bioactivation of cinnamic alcohol in a reconstructed human epidermis model and evaluation of sensitizing potency of the identified metabolites.

Background: Cinnamic alcohol is a natural compound, widely used in fragrances, which can cause allergic contact dermatitis. Cinnamic alcohol lacks intrinsic reactivity and autoxidation or metabolic activation is necessary for it to act as a sensitizer.

Methods: Bioactivation of cinnamic alcohol was explored using human liver microsomes, human liver S9 and SkinEthic™ Reconstructed Human Epidermis.

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.

Use of the TD50 99 % CI for single dose rodent carcinogenicity studies.

The Tumorigenic dose 50 (TD) is a widely used measure of carcinogenic potency representing the dosage at which 50 % of animals exposed to a compound will develop tumours. The popularity of the TD is in part due to the large amount of publicly available data. TDs were calculated for a large number of compounds in the Carcinogenic Potency Database, which has since been extended in the freely available Lhasa Carcinogenicity database, containing TDs from over 7500 studies and 1700 compounds.

Nitrosamine acceptable intakes should consider variation in molecular weight: The implication of stoichiometric DNA damage.

N-nitrosamines (NAs) are a class of compounds of which many, especially of the small dialkyl type, are indirect acting DNA alkylating mutagens. Their presence in pharmaceuticals is subject to very strict acceptable daily intake (AI) limits, which are traditionally expressed on a mass basis. Here we demonstrate that AIs that are not experimentally derived for a specific compound, but via statistical extrapolation or read across to a suitable analog, should be expressed on a molar scale or corrected for the target substance's molecular weight.

Revisiting the Landscape of Potential Small and Drug Substance Related Nitrosamines in Pharmaceuticals.

N-Nitrosamines are a class of indirect acting mutagens, as their metabolic degradation leads to the formation of the DNA-alkylating diazonium ion. Following up on the in-silico identification of thousands of nitrosamines that can potentially be derived from small molecule drugs and their known impurities described in a previous publication, we have now re-analyzed this dataset to apply EMA's Carcinogenic Potency Categorization Approach (CPCA) introduced with the 16th revision of their Q&A document for Marketing Authorization Holders. We find that the majority of potential nitrosamines from secondary amine precursors belongs to potency categories 4 and 5, corresponding to an acceptable daily intake of 1500 ng, whereas nitrosamines from tertiary amine precursors distribute more evenly among all categories, resulting in a substantial number of structures that are assigned the more challenging acceptable intakes of 18 ng/day and 100 ng/day for potency categories 1 and 2, respectively.

A deep dive into historical Ames study data for N-nitrosamine compounds.

Mutagenicity data is a core component of the safety assessment data required by regulatory agencies for acceptance of new drug compounds, with the OECD-471 bacterial reverse mutation (Ames) assay most widely used as a primary screen to assess drug impurities for potential mutagenic risk. N-Nitrosamines are highly potent mutagenic carcinogens in rodent bioassays and their recent detection as impurities in pharmaceutical products has sparked increased interest in their safety assessment. Previous literature reports indicated that the Ames test might not be sensitive enough to detect the mutagenic potential of N-nitrosamines in order to accurately predict a risk of carcinogenicity.

Maximizing use of existing carcinogenicity data to support acceptable intake levels for mutagenic impurities in pharmaceuticals: Learnings from N-nitrosamine case studies.

The unexpected finding of N-nitrosamine (NA) impurities in many pharmaceutical products raised significant challenges for industry and regulators. In addition to well-studied small molecular weight NAs, many of which are potent rodent carcinogens, novel NAs associated with active pharmaceutical ingredients have been found, many of which have limited or no safety data. A tiered approach to establishing Acceptable Intake (AI) limits for NA impurities has been established using chemical-specific data, read-across, or a class-specific TTC limit.

Acceptable intakes (AIs) for 11 small molecule N-nitrosamines (NAs).

Low levels of N-nitrosamines (NAs) were detected in pharmaceuticals and, as a result, health authorities (HAs) have published acceptable intakes (AIs) in pharmaceuticals to limit potential carcinogenic risk. The rationales behind the AIs have not been provided to understand the process for selecting a TD or read-across analog. In this manuscript we evaluated the toxicity data for eleven common NAs in a comprehensive and transparent process consistent with ICH M7.

Predicting Regioselectivity of Cytosolic Sulfotransferase Metabolism for Drugs.

Cytosolic sulfotransferases (SULTs) are a family of enzymes responsible for the sulfation of small endogenous and exogenous compounds. SULTs contribute to the conjugation phase of metabolism and share substrates with the uridine 5'-diphospho-glucuronosyltransferase (UGT) family of enzymes. UGTs are considered to be the most important enzymes in the conjugation phase, and SULTs are an auxiliary enzyme system to them.

Nature-Derived Epoxy Resin Monomers with Reduced Sensitizing Capacity─Isosorbide-Based Bis-Epoxides.

Epoxy resin systems (ERSs) are a class of thermosetting resins that become thermostable and insoluble polymers upon curing. They are widely used as components of protective surfaces, adhesives, and paints and in the manufacturing of composites in the plastics industry. The diglycidyl ether of bisphenol A (DGEBA) is used in 75-90% of ERSs and is thus by far the most used epoxy resin monomer (ERM).

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.

The Landscape of Potential Small and Drug Substance Related Nitrosamines in Pharmaceuticals.

This article reports the outcome of an in silico analysis of more than 12,000 small molecule drugs and drug impurities, identifying the nitrosatable structures, assessing their potential to form nitrosamines under relevant conditions and the challenges to determine compound-specific AIs based on data available or read-across approaches for these nitrosamines and their acceptance by health authorities. Our data indicate that the presence of nitrosamines in pharmaceuticals is likely more prevalent than originally expected. In total, 40.

What Makes a Potent Nitrosamine? Statistical Validation of Expert-Derived Structure-Activity Relationships.

The discovery of carcinogenic nitrosamine impurities above the safe limits in pharmaceuticals has led to an urgent need to develop methods for extending structure-activity relationship (SAR) analyses from relatively limited datasets, while the level of confidence required in that SAR indicates that there is significant value in investigating the effect of individual substructural features in a statistically robust manner. This is a challenging exercise to perform on a small dataset, since in practice, compounds contain a mixture of different features, which may confound both expert SAR and statistical quantitative structure-activity relationship (QSAR) methods. Isolating the effects of a single structural feature is made difficult due to the confounding effects of other functionality as well as issues relating to determining statistical significance in cases of concurrent statistical tests of a large number of potential variables with a small dataset; a naïve QSAR model does not predict any features to be significant after correction for multiple testing.

Predicting Regioselectivity of AO, CYP, FMO, and UGT Metabolism Using Quantum Mechanical Simulations and Machine Learning.

Unexpected metabolism in modification and conjugation phases can lead to the failure of many late-stage drug candidates or even withdrawal of approved drugs. Thus, it is critical to predict the sites of metabolism (SoM) for enzymes, which interact with drug-like molecules, in the early stages of the research. This study presents methods for predicting the isoform-specific metabolism for human AOs, FMOs, and UGTs and general CYP metabolism for preclinical species.

Updating the Dermal Sensitisation Thresholds using an expanded dataset and an in silico expert system.

The Dermal Sensitisation Thresholds (DST) are Thresholds of Toxicological Concern, which can be used to justify exposure-based waiving when conducting a skin sensitisation risk assessment. This study aimed to update the published DST values by expanding the size of the Local Lymph Node Assay dataset upon which they are based, whilst assigning chemical reactivity using an in silico expert system (Derek Nexus). The potency values within the expanded dataset fitted a similar gamma distribution to that observed for the original dataset.

Use of Lhasa Limited Products for the In Silico Prediction of Drug Toxicity.

Lhasa Limited have had a role in the in silico prediction of drug and other chemical toxicity for over 30 years. This role has always been multifaceted, both as a provider of predictive software such as Derek Nexus, and as an honest broker for the sharing of proprietary chemical and toxicity data. A changing regulatory environment and the drive for the Replacement, Reduction and Refinement (the 3Rs) of animal testing have led both to increased acceptance of in silico predictions and a desire for the sharing of data to reduce duplicate testing.

Developing Structure-Activity Relationships for -Nitrosamine Activity.

The detection of -nitrosodimethylamine (NDMA) in several marketed drugs led regulatory agencies to require that -nitrosamine risk assessments be performed on all marketed medical products [EMA/351053/2019 rev 1 (2019)]. Regulation of -nitrosamine impurity levels in pharmaceutical drug substances and products is described in the ICH M7(R1) guideline where they are referred to as "cohort-of-concern" compounds as several are potent rodent carcinogens [Kroes . 2004].

Use of less-than-lifetime (LTL) durational limits for nitrosamines: Case study of N-Nitrosodiethylamine (NDEA).

The ICH M7(R1) guideline describes a framework to assess the carcinogenic risk of mutagenic and carcinogenic pharmaceutical impurities following less-than-lifetime (LTL) exposures. This LTL framework is important as many pharmaceuticals are not administered for a patient's lifetime and as clinical trials typically involve LTL exposures. While there has been regulatory caution about applying LTL concepts to cohort of concern (COC) impurities such as N-nitrosamines, ICH M7 does not preclude this and indeed literature data suggests that the LTL framework will be protective of patient safety for N-nitrosamines.

Utilisation of parametric methods to improve percentile-based estimates for the carcinogenic potency of nitrosamines.

N-Nitrosamines have recently been the subject of intense regulatory scrutiny, including the setting of low exposure limits (18 ng/day) (European Medicines Agency (EMA), 2020). This paper evaluates different methodologies to determine statistically robust bounds on the carcinogenic potency of chemical classes, using historic TD data (Peto et al., 1984; Thresher et al.

Machine Learning Predicts Degree of Aromaticity from Structural Fingerprints.

Prediction of whether a compound is "aromatic" is at first glance a relatively simple task-does it obey Hückel's rule (planar cyclic π-system with 4n + 2 electrons) or not? However, aromaticity is far from a binary property, and there are distinct variations in the chemical and biological behavior of different systems which obey Hückel's rule and are thus classified as aromatic. To that end, the aromaticity of each molecule in a large public dataset was quantified by an extension of the work of Raczyńska et al. Building on this data, a method is proposed for machine learning the degree of aromaticity of each aromatic ring in a molecule.