Krista L. Dobo

Use of in silico systems and expert knowledge for structure-based assessment of potentially mutagenic impurities

Genotoxicity hazard identification is part of the impurity qualification process for drug substances and products, the first step of which being the prediction of their potential DNA reactivity using in silico (quantitative) structure-activity relationship (Q)SAR models/systems. This white paper provides information relevant to the development of the draft harmonized tripartite guideline ICH M7 on potentially DNA-reactive/mutagenic impurities in pharmaceuticals and their application in practice. It explains relevant (Q)SAR methodologies as well as the added value of expert knowledge. Moreover, the predictive value of the different methodologies analyzed in two surveys conveyed in the US and European pharmaceutical industry is compared: most pharmaceutical companies used a rule-based expert system as their primary methodology, yielding negative predictivity values of ⩾78% in all participating companies. A further increase (>90%) was often achieved by an additional expert review and/or a second QSAR methodology. Also in the latter case, an expert review was mandatory, especially when conflicting results were obtained. Based on the available data, we concluded that a rule-based expert system complemented by either expert knowledge or a second (Q)SAR model is appropriate. A maximal transparency of the assessment process (e.g. methods, results, arguments of weight-of-evidence approach) achieved by e.g. data sharing initiatives and the use of standards for reporting will enable regulators to fully understand the results of the analysis. Overall, the procedures presented here for structure-based assessment are considered appropriate for regulatory submissions in the scope of ICH M7.

In silico methods combined with expert knowledge rule out mutagenic potential of pharmaceutical impurities: an industry survey.

With the increasing emphasis on identification and low level control of potentially genotoxic impurities (GTIs), there has been increased use of structure-based assessments including application of computerized models. To date many publications have focused on the ability of computational models, either individually or in combination, to accurately predict the mutagenic effects of a chemical in the Ames assay. Typically, these investigations take large numbers of compounds and use in silico tools to predict their activity with no human interpretation being made. However, this does not reflect how these assessments are conducted in practice across the pharmaceutical industry. Current guidelines indicate that a structural assessment is sufficient to conclude that an impurity is non-mutagenic. To assess how confident we can be in identifying non-mutagenic structures, eight companies were surveyed for their success rate. The Negative Predictive Value (NPV) of the in silico approaches was 94%. When human interpretation of in silico model predictions was conducted, the NPV increased substantially to 99%. The survey illustrates the importance of expert interpretation of in silico predictions. The survey also suggests the use of multiple computational models is not a significant factor in the success of these approaches with respect to NPV.

Defining EMS and ENU dose-response relationships using the Pig-a mutation assay in rats.

In recent years, experimental evidence has accumulated that supports the existence of sublinear dose-response relationships at low doses of DNA reactive mutagens. However, creating the in vivo data necessary to allow for a more detailed dose-response modeling with the currently available tools might not always be practical. The purpose of the current work was to evaluate the utility of the Pig-a gene mutation assay to rapidly identify dose-response relationships for direct acting genotoxicants. The induction of mutations in the peripheral blood of rats was evaluated following 28 days of exposure down to low doses of the direct acting alkylating agents ethyl methane sulfonate (EMS) and ethylnitrosourea (ENU). Using statistical modeling based on the 28-day studies, a threshold for mutation induction for EMS was estimated to be 21.9mg/kg, whereas for the more potent ENU, the threshold was estimated to be 0.88mg/kg. Comparing mutation frequencies from acute and sub-chronic dosing indicated less than additive dose-response relationships, further confirming the possibility of a threshold dose-response relationship for both compounds. In conclusion, the work presented provides evidence that the Pig-a assay might be a practical alternative to other in vivo mutation assays when assessing dose-response relationships for direct acting mutagens and that an experimental approach using fractionated dosing could be used to substantiate a biological mechanism responsible for the observation of a sublinear dose-response relationship.

Report on stage III Pig-a mutation assays using N-ethyl-N-nitrosourea – comparison with other in vivo genotoxicity endpoints

N‐Ethyl‐N‐nitrosourea (ENU) was evaluated as part of the Stage III trial for the rat Pig‐a gene mutation assay. Groups of six‐ to eight‐week‐old male Sprague Dawley (SD) or Fischer 344 (F344) rats were given 28 daily doses of the phosphate buffered saline vehicle, or 2.5, 5, or 10 mg/kg ENU, and evaluated for a variety of genotoxicity endpoints in peripheral blood, spleen, liver, and colon. Blood was sampled predose (Day‐1) and at various time points up to Day 57. Pig‐a mutant frequencies were determined in total red blood cells (RBCs) and reticulocytes (RETs) as RBCCD59− and RETCD59− frequencies. Consistent with the results from a reference laboratory, RBCCD59− and RETCD59− frequencies increased in a dose‐ and time‐dependent manner, producing significant increases at all doses by Day 15, with similar frequencies seen in both rat strains. ENU also induced small but significant increases in % micronucleated RETs on Days 4 and 29. No significant increases in micronuclei were seen in the liver or colon of the ENU‐treated SD rats. Hprt and Pig‐a lymphocyte mutation assays conducted on splenocytes from Day 56 F344 rats detected two‐ to fourfold stronger responses for Hprt than Pig‐a mutations. Results from the in vivo Comet assay in SD rats at Day 29 showed generally weak increases in DNA damage in all tissues evaluated. The results with ENU indicate that the Pig‐a RET and RBC assays are reproducible, transferable, and complement other genotoxicity endpoints that could potentially be integrated into 28‐day repeat‐dose rat studies.

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.

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. This may be followed by an assessment of additional information that serves as the basis for an expert review to support or refute the predictions. This paper elucidates scenarios where additional expert knowledge may be beneficial, what such an expert review may contain, and how the results and accompanying considerations may be documented. Furthermore, the use of these principles and procedures to yield a consistent and robust (Q)SAR-based argument to support impurity qualification for regulatory purposes is described in this manuscript.

A Strategy for the Risk Assessment of Human Genotoxic Metabolites

The role of metabolism in genotoxicity and carcinogenicity of many chemicals is well established. Accordingly, both in vitro metabolic activation systems and in vivo assays are routinely utilized for genotoxic hazard identification of drug candidates prior to clinical investigations. This should, in most cases provide a high degree of confidence that the genotoxic potential of the parent and associated metabolites have been characterized. However, it is well known that significant differences can exist between human metabolism and that which occurs with in vitro and in vivo genotoxicity tests. This poses challenges when considering the adequacy of hazard identification and cancer risk assessment if a human metabolite of genotoxic concern is identified during the course of drug development. Since such challenges are particularly problematic when recognized in the later stages of drug development, a framework for conducting a carcinogenic risk assessment for human genotoxic metabolites is desirable. Here, we propose a risk assessment method that is dependent upon the availability of quantitative human and rodent ADME (absorption, distribution, metabolism, excretion) data, such that exposures to a metabolite of genotoxic concern can be estimated at the intended human efficacious dose and the maximum dose used in the 2-year rodent bioassay(s). The exposures are then applied to the risk assessment framework, based on known cancer potencies, that allows one to understand the probability of a known or suspect genotoxic metabolite posing a carcinogenic risk in excess of 1 in 100,000. Practical case examples are presented to illustrate the application of the risk assessment method within the context of drug development and to highlight its utility and limitations.

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. In addition, the guidance states that the output from these computer-based assessments can be reviewed using expert knowledge to provide additional support or resolve conflicting predictions. This approach is designed to maximize the sensitivity for correctly identifying DNA reactive compounds while providing a framework to reduce the number of compounds that need to be synthesized, purified and subsequently tested in an Ames assay. Using a data set of 801 chemicals and pharmaceutical intermediates, we have examined the relative predictive performances of some popular commercial in silico systems that are in common use across the pharmaceutical industry. The overall accuracy of each of these systems was fairly comparable ranging from 68% to 73%; however, the sensitivity of each system (i.e. how many Ames positive compounds are correctly identified) varied much more dramatically from 48% to 68%. We have explored how these systems can be combined under the ICH M7 guidance to enhance the detection of DNA reactive molecules. Finally, using four smaller sets of molecules, we have explored the value of expert knowledge in the review process, especially in cases where the two systems disagreed on their predictions, and the need for care when evaluating the predictions for large data sets.

Comparison of metabolite profiles generated in Aroclor‐induced rat liver and human liver subcellular fractions: Considerations for in vitro genotoxicity hazard assessment

Because it is well known that metabolites of chemicals and drugs are frequently the ultimate species responsible for genotoxicity and carcinogenicity, in vitro testing to identify the human genotoxicity hazard potential of new chemicals and drugs routinely utilizes liver S‐9 fraction from rats treated with Aroclor 1254 as a system that can generate metabolites. However, it is frequently questioned as to whether such an in vitro metabolite generation system is the most relevant for human risk, or whether the assay would be better served by using a human‐derived in vitro system. To address this, 16 common drugs have been examined for profiles of metabolites in Aroclor‐induced rat liver S‐9 and pooled human liver S‐9. Metabolite profiles were compared using high pressure liquid chromatography coupled with ion trap mass spectrometry, in line with ultraviolet or radiometric detection to help make semiquantitative comparisons. Results showed that, with few exceptions, metabolites generated in the human system were also generated in the rat system. Also, in several cases the rat system generated considerably more metabolites, suggesting that there is a potential that positive genotoxicity findings could be caused by metabolites that have no relevance to humans. These findings suggest that when conducting in vitro genotoxicity testing using the Aroclor‐induced rat liver S‐9 system, knowledge of the metabolite profile in the system is important, and a comparison to the profile generated in human liver S‐9 could be of value when interpreting the genotoxicity results. Environ. Mol. Mutagen., 2008.

Overview of Genotoxic Impurities in Pharmaceutical Development

This symposium focuses on the management of genotoxic impurities in the synthesis of pharmaceuticals. Recent developments in both Europe and United States require sponsors of new drug applications to develop processes to control the risks of potential genotoxic impurities. Genotoxic impurities represent a special case relative to the International Conference on Harmonisation Q3A/Q3B guidances, because genotoxicity tests used to qualify the drug substance may not be sufficient to demonstrate safety of a potentially genotoxic impurity. The default risk management approach for a genotoxic impurity is the threshold of toxicological concern unless a more specific risk characterization is appropriate. The symposium includes descriptions of industry examples where impurities are introduced and managed in the synthesis of a pharmaceutical. It includes recent regulatory developments such as the “staged threshold of toxicological concern” when administration is of short duration (eg, during clinical trials).