Mutagenic & Genotoxic Assessment within Seqens’ Lab Ecosystem
The synthesis of drug substances involves the use of chemicals, reagents, solvents, catalysts, and other processing aids. As a result, the importance to fully understand and control the chemical synthesis and subsequent degradation to impurities are compulsory during the development of drug products. Analytical excellence is therefore essential for ensuring quality control.
Mutagenicity and Genotoxicity hazard identification are part of the impurity qualification process for drug substances and products. For clarification, Mutagenicity refers to the production of transmissible genetic alterations, as Genotoxicity covers a broader spectrum of endpoints than mutagenicity, including DNA damage assessments. DNA damage are not themselves necessarily transmissible to the next generation of cells, they may be mutagens.
Current methods used in mutagenicity and genetic toxicity assessment, are in silico approaches and several in vitro (e.g. Ames test) or in vivo assays.
The in vitro tests are suitable for assessing primary genotoxicity, whereas in vivo approach can provide information about secondary genotoxic effects via inflammation, oxidative stress mostly. These tests have their own endpoints and specificities to examine the genotoxic activity caused by various toxic substances on biologically different levels.
The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) M7 guideline for the assessment and control of mutagenic impurities in pharmaceuticals was established in 2014. This international guideline[i] has fully approved to submit data according to structure-based assessment by using in silico quantitative structure-activity relationship (QSAR) models. It provides relevant information on potentially DNA-reactive/mutagenic impurities in pharmaceuticals and their application in practice. Therefore, in the absence of experimental data, QSAR models are considered as reliable predictive tools for identifying mutagenic chemicals.
As Seqens Lab’s partner, Hamonic Pharma?provide SAFETY BY DESIGN?, ?an innovative in silico services and software that benefit the regulatory file, guidelines for studies In human and the alternative to animal testing. SAFETY BY DESIGN? is used to investigate the potential genotoxicity of active pharmaceutical ingredient (API) derived impurities / intermediates with two complementary methodologies required by the ICH M7 regulation – i.e. statistical-based and expert rule-based. The corresponding investigation report is fully validated by a toxicology expert approved by the European Register of Toxicologists (ERT).
In vitro test: Ames / Nano-Ames
The Ames test[ii] (the bacterial reverse mutation assay), developed in the 1970s by Bruce Ames, is the widely used in vitro mutagenesis test. The assay is carried out using different specific Salmonella typhimurium and Escherichia coli strains carrying a defective (mutant) gene, which inhibits expression of the essential amino acid. The test is based on inducing reverse mutations in the defective gene.
Mutagenic agents which cause reverse mutation at the defective gene will enable the bacteria to synthesize the essential amino acid (such as histidine or tryptophan) and grow as a visible colony when plated in minimal media. The number of these colonies is a direct measure of the mutagenicity of the test substance in the individual strains, which also enables conclusions with respect to the underlying mechanism of mutation induction.
Noteworthy, the miniaturized Ames test is also widely used for screening approach[iii] or in genotoxicity (GTI) strategy by pharmaceutical companies. Indeed, the major limitation of the Ames test is the needed quantity of compound to be tested, around at least 300 mg by experiment in regulatory Ames test and 20 mg for the miniaturized Ames test.
GenEvolutioN is a GLP compliance CRO for drugs, cosmetics, chemicals and medical devices dedicated to in vitro toxicology and in vitro genetic toxicology. The company has developed Nano-Ames test (NanoAmesTM), which is an ultra-miniaturized method. It is an adaptation of the regular Ames test in order to reduce the needed quantity of compound by retaining the design of the regulatory studies: 5 strains with and without metabolic activation. In these tests, micrograms to nanograms are only required. It is the appropriate test for traces of impurity characterization. Only 0,1 mg of test sample is required for the 5 OECD strains.
In silico approaches:
The ICH M7 guideline on Drug impurities recognizes the ability of QSAR models to relate chemical structure and bacterial mutagenicity (Ames test). Those computational methods can predict the potential adverse effects of a chemical based on its chemical structure and properties.
In the field of mutagenicity prediction of chemical substances QSAR models are the most advanced ones, and the approach has already been in practical use for several years[iv].
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Statistical-based QSAR uses molecular descriptors (numerical data) such as geometric, electronic, physicochemical, and descriptors that correlate highly with positive results in the Ames test. A selection of descriptors are then processed through a machine learning method to constitute a model able to investigate the potential mutagenicity of a given chemical.
Results from the Ames test show a high reproducibility rate (80–85%) and are consistent in comparison with results from other toxicological tests5. In addition, the Ames test has generated the greatest quantity of accumulated toxicity data. In this perspective, that test is of particular interest and relevant to develop a robust QSAR mutagenicity prediction model.
Concluding remarks
As we move away from preclinical animal testing, there is a growing demand for developing reliable genetic toxicology methods. Altogether the accumulated experimental data that have been gathered for decades have allowed these aforementioned in vitro and in silico methods to become extremely powerful tools to deliver robust results with regard to the investigation of early toxicity of chemicals at strategic stages in a reasonable time scale.
To address these challenges, an integrated approach which combines the key competences for impurities characterization will benefit from this virtuous proximity as we share the same location :
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[i] ICH-M7 (R1); ICH Harmonized Guideline. Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk. Current Step 4, version dated 31 March 2017. https:// www.ich.org/home.html.
[ii] Ames BN, McCann J and Yamasaki E, Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutation Research, 1975, 31,347-364
[iii] Burke DA, Wedd DJ and Burlinson B. Use of the Miniscreen assay to screen novel compounds for bacterial mutagenicity in the pharmaceutical industry, Mutagenesis, 1996, vol.11, 2, 201-205.
[iv] The Organisation for Economic Cooperation and Development (OECD) indicated that QSAR will be used actively in future toxicity assessments of chemical substances and published principles for international harmonization in relation to QSAR assessment.
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1 年Juliette Martin Frédéric Schab Gautier Decock