In silico modelling of microbial and human metabolism: a case study with the fungicide carbendazim

A major source of uncertainty when assessing the human health and environmental risks of chemicals is the paucity of experimental information on the metabolic and (bio)degradation pathways of parent compounds and the toxicological properties of their metabolites and (bio)degradation products. Taking into account animal welfare and cost-effectiveness considerations, the only practical means of obtaining the information needed to reduce this uncertainty, is to use alternative (non-animal) methods, such as in vitro tests and in silico models. In this report, we explore the usefulness of in silico metabolic simulation tools (expert systems) as a means of supporting the regulatory assessment of chemicals. In particular, we investigate the use of selected in silico tools to: (i) simulate microbial and mammalian metabolic pathways; (ii) identify potential metabolites resulting from biotransformation; and (iii) gain insights into the mechanistic rationale of simulated metabolic reactions and the likelihood of their occurrence. For illustrative purposes, the microbial and mammalian biotransformation pathways of a case study compound, the fungicide carbendazim, were generated by using the CRAFT Explorer 1.0 (Molecular Networks GmbH) and Meteor 12.0.0 (Lhasa Ltd.) software tools. Additionally, the set of potential metabolites resulting from microbial and mammalian metabolism was predicted with the OECD QSAR Application Toolbox 2.0 (beta version). Comparison of the in silico predictions with existing experimental data on carbendazim metabolism showed the potential usefulness of using software tools for metabolite prediction. However, the results are strongly dependent on the software constraints specified by the user, and require careful interpretation, taking into account the needs of the exercise and the availability of existing information. Further efforts are needed to develop guidance on the use of in silico metabolic simulation tools for the purposes of regulatory risk assessments. LIST OF ABBREVIATIONS 2-AB 2-Aminobenzimidazole 5,6-DHCB Methyl (5,6-dihydroxy-1H-benzimidazol-2-yl)carbamate 5,6-DHCB-G Glucuronic acid conjugate of methyl (5,6-dihydroxy-1Hbenzimidazol2-yl)carbamate 5,6-DHCB-S Sulphuric acid conjugate of Methyl (5,6-dihydroxy-1Hbenzimidazol-2-yl)carbamate 5,6-DHHBC-G S-[5,6-dihydro-5-hydroxy-2-(methoxycarbonylamino)-1Hbenzimidazol-6-yl]glutathione 5,6-HOBC N-oxide Methyl (5-hydroxy-6-oxo-6H-benzimidazol-2-yl)carbamate N1oxide 5,6-HOBC N-oxide-G Glucuronide of Methyl (5-hydroxy-6-oxo-6H-benzimidazol-2yl)carbamate N1-oxide 5-HCB Methyl 5-hydroxy-1H-benzimidazol-2-ylcarbamate 5-HCB-G Glucuronic acid conjugate of Methyl 5-hydroxy-1H-benzimidazol-2ylcarbamate 5-HCB-S Sulphuric acid conjugate of Methyl 5-hydroxy-1H-benzimidazol-2ylcarbamate AGES Austrian Agency for Health and Food Safety Bt Biotransformation CAS No Chemical Abstracts Service Registry Number CRAFT Chemical Reactivity and Fate Tool CYP450 Cytochrome P450 EC European Commission EEC European Economic Community EFSA European Food Safety Authority EU European Union FAO Food and Agriculture Organisation IUPAC International Union of Pure and Applied Chemistry JMPR Joint FAO/WHO Meeting on Pesticide Residues JRC Joint Research Centre (of the European Commission) Log P Octanol/Water partition coefficient M Meteor 12.0.0 Metabolite OECD Organisation for Economic Cooperation and Development P CRAFT Explorer 1.0 (Bio)Degradation Product PPP Plant Protection Product Q Meteor 12.0.0 Query Compound QSAR Quantitative Structure-Activity Relationship SA Structural Alert SAR Structure-Activity Relationship SMILES Simplified Molecular Input Line Entry Specification TIMES Tissue Metabolism Simulator TTC Threshold of Toxicological Concern UGT UDP-glucuronosyltransferase UM-BBD University of Minnesota Biocatalysis and Biodegradation Database WHO World Health Organisation