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

A thorough literature review was undertaken to understand how the pathways of N-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.g., ß-carbon), and other reactive species can form following initial bioactivation, although these alternative pathways tend to decrease rather than enhance carcinogenic potency. The activation pathway, oxidative dealkylation, is a common reaction in drug metabolism and evidence indicates that the carbonyl byproduct, e.g., formaldehyde, does not contribute to the toxic properties of N-nitrosamines. Nitric oxide (NO), a side product of denitrosation, can similarly be discounted as an enhancer of N-nitrosamine toxicity based on carcinogenicity data for substances that act as NO-donors. However, not all N-nitrosamines are potent rodent carcinogens. In a significant number of cases, there is a potency overlap with non-N-nitrosamine carcinogens that are not in the Cohort of Concern (CoC; high-potency rodent carcinogens comprising aflatoxin-like-, N-nitroso-, and alkyl-azoxy compounds), while other N-nitrosamines are devoid of carcinogenic potential. In this context, mutagenicity is a useful surrogate for carcinogenicity, as proposed in the ICH M7 (R2) (2023) guidance. Thus, in the safety assessment and control of N-nitrosamines in medicines, it is important to understand those complementary attributes of mechanisms of mutagenicity and structure-activity relationships that translate to elevated potency versus those which are associated with a reduction in, or absence of, carcinogenic potency.

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 TD50 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. This evaluation included substances which had datasets that were robust, limited but sufficient, and substances with insufficient experimental animal carcinogenicity data. In the case of robust or limited but sufficient carcinogenicity information, AIs were calculated based on published or derived TD50s from the most sensitive organ site. In the case of insufficient carcinogenicity information, available carcinogenicity data and structure activity relationships (SARs) were applied to categorical-based AIs of 1500 ng/day, 150 ng/day or 18 ng/day; however additional data (such as biological or additional computational modelling) could inform an alternative AI. This approach advances the methodology used to derive AIs for NAs.

Mutagenic impurities in pharmaceuticals: A critical assessment of the cohort of concern with a focus on N-nitrosamines.

The TTC (Threshold of Toxicological Concern; set at 1.5 µg/day for pharmaceuticals) defines an acceptable patient intake for any unstudied chemical posing a negligible risk of carcinogenicity or other toxic effects. A group of high potency mutagenic carcinogens, defined solely by the presence of particular structural alerts, are referred to as the "cohort of concern" (CoC); aflatoxin-like-, N-nitroso-, and alkyl-azoxy compounds are considered to pose a significant carcinogenic risk at intakes below the TTC. Kroes et al. (2004) derived values for the TTC and CoC in the context of food components, employing a non-transparent dataset never placed in the public domain. Using a reconstructed all-carcinogen dataset from relevant publications, it is now clear that there are exceptions for all three CoC structural classes. N-Nitrosamines represent 62% of the N-nitroso class in the reconstructed dataset. Employing a contemporary dataset, 20% are negative in rodent carcinogenicity bioassays with less than 50% of all N-nitrosamines estimated to fall into the highest risk category. It is recommended that CoC nitrosamines are identified by compound-specific data rather than structural alerts. Thus, it should be possible to distinguish CoC from non-CoC N-nitrosamines in the context of mutagenic impurities described in ICH M7 (R1).

Tolerability of risk: A commentary on the nitrosamine contamination issue.

For decades, regulators have grappled with different approaches to address the issue of control of impurities. Safety-based limits, such as permissible daily exposure (PDE), acceptable intake (AI), threshold of toxicological concern (TTC) and less than lifetime limits (LTL) have all been used. For many years these safety-based limits have been recognized as virtually safe doses (VSDs). Recently, however, many regulatory agencies are seeking to impose limits for N-nitrosamine impurities, which are significantly below the VSD. This commentary will discuss the evolution of safety-based limits for impurities, provide an overview of the valsartan N-nitrosamine contamination issue and review the toxicology of N-nitrosamines. The outcome of a lessons-learned exercise on sartan medications undertaken by the European Medicines Agency (EMA) will also be discussed. The review will also highlight the many analytical challenges inherent with controlling impurities to ppb-based limits. The use of highly sensitive, low ppb limits, methods may lead to future issues of batch rejection, based on false positives. Regulators initially viewed the N-nitrosamine risk as being insufficient to prompt immediate product discontinuation and patients were specifically advised to continue using their affected medication. Patients were also informed that exposure to N-nitrosamines is extremely common via food and drinking water.

Short commentary on NDMA (N-nitrosodimethylamine) contamination of valsartan products.

A range of generic valsartan products have been found to be contaminated with nitrosamines (principally N-nitrosodimethylamine; NDMA). We present information and discuss various elements of this phenomenon including: actions taken by regulatory agencies, source of the nitrosamine impurities, range of possible risk assessments based mainly on ICH M7 criteria, epidemiological assessment and analytical aspects.

Extending (Q)SARs to incorporate proprietary knowledge for regulatory purposes: is aromatic N-oxide a structural alert for predicting DNA-reactive mutagenicity?

(Quantitative) structure-activity relationship or (Q)SAR predictions of DNA-reactive mutagenicity are important to support both the design of new chemicals and the assessment of impurities, degradants, metabolites, extractables and leachables, as well as existing chemicals. Aromatic N-oxides represent a class of compounds that are often considered alerting for mutagenicity yet the scientific rationale of this structural alert is not clear and has been questioned. Because aromatic N-oxide-containing compounds may be encountered as impurities, degradants and metabolites, it is important to accurately predict mutagenicity of this chemical class. This article analysed a series of publicly available aromatic N-oxide data in search of supporting information. The article also used a previously developed structure-activity relationship (SAR) fingerprint methodology where a series of aromatic N-oxide substructures was generated and matched against public and proprietary databases, including pharmaceutical data. An assessment of the number of mutagenic and non-mutagenic compounds matching each substructure across all sources was used to understand whether the general class or any specific subclasses appear to lead to mutagenicity. This analysis resulted in a downgrade of the general aromatic N-oxide alert. However, it was determined there were enough public and proprietary data to assign the quindioxin and related chemicals as well as benzo[c][1,2,5]oxadiazole 1-oxide subclasses as alerts. The overall results of this analysis were incorporated into Leadscope's expert-rule-based model to enhance its predictive accuracy.

Mutagenic impurities in pharmaceuticals: a critique of the derivation of the cancer TTC (Threshold of Toxicological Concern) and recommendations for structural-class-based limits.

The cancer TTC (Threshold of Toxicological Concern) concept is currently employed as an aid to risk assessment of potentially mutagenic impurities (PMIs) in food, cosmetics and other sectors. Within the pharmaceutical industry the use of one default cancer TTC limit of 1.5 µg/day for PMIs is being increasingly questioned. Its derivation, originally in the context of foodstuffs, can be broken down into five key elements: dataset composition; determination of carcinogenicity/mutagenicity status and carcinogenic potency (based on TD50s) of compounds in the dataset; linear extrapolation of carcinogenic potencies; evaluation of the more potent compounds in each structural category, and presence of representative structural alerts amongst the more potent compounds. A detailed evaluation reveals that the derivation process is distorted by the use of the lowest statistically significant TD50s (which can produce a false-carcinogen phenomenon) and by employing linear extrapolation for non-mutagenic carcinogens. By correcting for these two factors, it is concluded that only around 50% of conventional structural-alert categories were adequately addressed and that limits higher than the default value appear to be justified in many cases. Using similar criteria for PMIs in pharmaceuticals, four distinct potency categories of conventional structural alerts can be distinguished, ranging from alerts with questionable validity to those with high potency, which are considered to provide a range of flexible and pragmatic limits for such impurities.

Guidelines and pharmacopoeial standards for pharmaceutical impurities: overview and critical assessment.

ICH/regional guidances and agency scrutiny provide the regulatory framework for safety assessment and control of impurities in small-molecule drug substances and drug products. We provide a critical assessment of the principal impurity guidances and, in particular, focus on deficiencies in the derivation of the threshold of toxicological concern (TTC) as applied to genotoxic impurities and the many toxicological anomalies generated by following the current guidelines on impurities. In terms of pharmacopoeial standards, we aim to highlight the fact that strictly controlling numerous impurities, especially those that are minor structural variants of the active substance, is likely to produce minimal improvements in drug safety. It is believed that, wherever possible, there is a need to simplify and rebalance the current impurity paradigm, moving away from standards derived largely from batch analytical data towards structure-based qualification thresholds and risk assessments using readily available safety data. Such changes should also lead to a minimization of in vivo testing for toxicological qualification purposes. Recent improvements in analytical techniques and performance have enabled the detection of ever smaller amounts of impurities with increased confidence. The temptation to translate this information directly to the regulatory sphere without any kind of safety evaluation should be resisted.

Drug substances presented as sulfonic acid salts: overview of utility, safety and regulation.

OBJECTIVES: Controlling genotoxic impurities represents a significant challenge to both industry and regulators. The potential for formation of genotoxic short-chain alkyl esters of sulfonic acids during synthesis of sulfonic acid salts is a long-standing regulatory concern. This review provides a general overview of the utility of sulfonic acids as salt-forming moieties and discusses strategies for effectively minimizing the potential for alkyl sulfonate formation during the synthesis and processing of sulfonate salt active pharmaceutical ingredients. The potential implications of the recent establishment of a substantial human threshold dose for ethyl methanesulfonate for the safety assessment of alkyl sulfonates in general are also discussed. KEY FINDINGS: The formation of alkyl sulfonates requires highly acidic conditions, possibly combined with long reaction times and/or elevated temperatures, to generate significant amounts, and these conditions are most unlikely to be present in the synthesis of active pharmaceutical ingredient sulfonate salts. It is possible to design salt formation conditions, using a short-chain alcohol as solvent, to manufacture sulfonate salts that are essentially free of alkyl sulfonate impurities. Processes using non-acidic conditions such as ethanol recrystallization or wet granulation should not raise any concerns of alkyl sulfonate formation. SUMMARY: An understanding of the mechanism of formation of alkyl sulfonates is critical in order to avoid restricting or over-controlling sulfonic acid salts, which have many technical advantages as pharmaceutical counterions. Recent regulatory acceptance of a human threshold limit dose of 2 mg/kg per day for ethyl methanesulfonate, indicating that its toxicological risks have previously been considerably overestimated, could signal the beginning of the end over safety concerns on alkyl sulfonate residues, thus removing a major constraint from the exploitation of sulfonic acid counterions.

Understanding and applying regulatory guidance on the nonclinical development of biotechnology-derived pharmaceuticals.

Biotechnology-derived pharmaceuticals are a well established and growing part of the therapeutic armamentarium. Beginning with recombinant versions of products such as insulin that were previously manufactured by extraction from animal and human sources, licensed biotechnology drugs and those in development now span an ever-increasing range of product types and therapeutic categories. As a consequence of this diversity, both general and product class-specific scientific guidelines have been developed on a regional (e.g. EU/US) or international (e.g. ICH - International Conference on Harmonization) basis. The current portfolio of nonclinical guidelines, particularly ICH S6, emphasizes flexibility and adaptability to the specific circumstances of the individual biotechnology product and its intended indication, taking into account factors not generally applicable to small-molecule drugs, such as pharmacodynamic responsiveness of safety and efficacy models, species specificity, and antibody formation. Guidelines developed principally with small-molecule drugs in mind may, nevertheless, have some applicability to biotechnology drugs on issues such as safety pharmacology, as well as on regulatory, procedural and dossier submission requirements. Scientific guidelines, such as those providing nonclinical guidance, are just one, albeit important, component of an increasingly complex legal/scientific environment in drug development.

Residues of genotoxic alkyl mesylates in mesylate salt drug substances: real or imaginary problems?

Mesylate esters of short-chain (n = 1-3) alcohols are reactive, direct-acting, genotoxic and possibly carcinogenic alkylating agents. Their chemical and biological properties appear to correlate well with Swain-Scott s constants; for example, high S(N)1 character (low s value) is associated with enhanced carcinogenic potential, but also a rapid hydrolysis rate. Concerns over the possible formation of such esters during the preparation of mesylate salt drug substances, by addition of methane sulfonic acid (MSA) to the free base dissolved in an alcoholic solvent, have led regulatory agencies to require applicants to demonstrate that the synthetic method employed does not lead to the presence of detectable levels of alkyl mesylates. Mechanistic considerations, relating mainly to the extremely low nucleophilicity of the mesylate anion, and experimental data, both indicate that alkyl mesylates should not be formed (except from MSA impurities) during mesylate salt synthesis. Mechanistic arguments also predict that residues of alkyl halides (possibly formed in the preparation of amine hydrochlorides or hydrobromides) could represent a similar or greater potential hazard than alkyl mesylates. The perceived risk of alkyl mesylate formation seems to rely on mistaken assumptions and so the concerns appear unjustified. Further reassurance could be achieved however by applying a variety of strategies during synthesis, including pH control, and use of high-purity MSA or of a non-hydroxylic reaction solvent.

Regulatory immunotoxicology: does the published evidence support mandatory nonclinical immune function screening in drug development?

Recent immunotoxicity guidance documents from the EU CHMP and the US FDA apply significantly different weightings to immune function testing; whereas the former mandates (as a starting point) incorporation of immune function tests (IFTs) to screen for immunotoxic potential in sub-chronic rodent toxicity studies, the more cautious 'for cause' FDA approach recommends the use of IFTs only when warranted by evidence obtained from conventional nonclinical and/or clinical studies. Conclusions from detailed evaluations of several key drugs, including salmeterol and some opioids, challenge the notion that data on these examples support the need for IFTs to detect unintended immunosuppression. Given the virtual absence of convincing pharmaceutical examples and the rarity of unintended immunosuppression, routine immune function testing of all new pharmaceuticals is not considered justified. Resources currently being employed in this manner in an attempt to detect a seemingly rare phenomenon would appear to be better applied to the development of reliable predictive assays for drug hypersensitivity, which is known to cause significant patient morbidity. Any moves towards a globally harmonised guideline that recommends the use of concern-based IFTs, need ideally to be accompanied by the establishment of appropriate historical control reference intervals and interpretation criteria to support a reliable weight-of-evidence approach to data evaluation.

An EU perspective on the use of in vitro methods in regulatory pharmaceutical toxicology.

Some in vitro methods such as those used in the assessment of genotoxicity, receptor-binding and QT-prolongation are well established in regulatory pharmaceutical toxicology. In vitro systems to study metabolic profiles, P450 isoforms, drug interactions, etc. or to provide metabolic activation in genotoxicity assays are extremely useful, but are subject to a number of important limitations. In vitro models are also employed on an ad-hoc basis for other purposes, for example, to help investigate mechanisms underlying in vivo findings. At the current stage of technical development of alternative methods, rapid replacement of the pivotal animal studies used in drug safety assessment seems unlikely. The existing in vivo models have good predictive ability regarding toxic effects in humans, are underpinned by an extensive literature and form the basis of most regulatory toxicology guidelines. Integrated in vitro testing strategies, meant to replace conventional repeated-dose studies, are still relatively undeveloped. Emerging technologies such as transgenics, toxicogenomics and toxicoproteomics, although they rely on the continued use of animals, have considerable potential in terms of reduction and refinement of in vivo methods.

Alternatives models in carcinogenicity testing--a European perspective.

Transgenic mouse strains offer the prospect of significant benefits in the in vivo assessment of carcinogenic potential. The European Regulatory Authorities have been supportive of their inclusion as one of the second-test options in the International Conference on Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human use (ICH). However, there is a concern regarding premature systematic use of these models. At present, the information from the International Life Sciences Institute (ILSI) project suggests that the transgenic models under study are similarly sensitive to genotoxic pharmaceuticals. There are apparently some false negatives and false positives. For regulatory purposes, it is not yet possible to differentiate the models with respect to hazard identification and risk assessment. The evaluation of the models has reached an interesting but, at certain points, equivocal stage. Based on the weight of evidence gathered thus far, regulatory authorities cannot neglect the outcome of such studies but need to be cautious in their interpretation of data from such models, and the application in risk assessment procedures.