Introduction A number of biological effects are brought about by the covalent interaction of a xenobiotic molecule, such as a drug, and a biological macromolecule. For example, a molecule reacting covalently with DNA may lead to mutagenesis, with an immunoprotein may lead to sensitisation, and with a skin cell may lead to irritation. These interactions can be inter- preted in terms of the (organic) chemistry mecha- nisms by which they proceed (1). If we can understand these interactions and capture the rele- vant information, we may then be able to utilise the knowledge to predict the possibility of toxicity with- out recourse to animal testing. This area of captur- ing knowledge relating to the intrinsic reactivity of a substance is termed “in chemico”, in line with in vivo, in vitro, and in silico. It is important to appre- ciate that these in chemico techniques should not be seen as being another in vitro approach, for a num- ber of reasons: they are physicochemical measure- ments and do not contain biological material (with the exception of some approaches that utilise enzyme fractions), in addition they should not be considered as a direct one-to-one replacement, as is often the case with in vitro techniques. The use of experimental measurements of in chemico reactivity has been the subject of a recent European Centre for the Validation of Alternative Methods (ECVAM) workshop (2). In the workshop report, the term in chemico is defined as referring to “the use of abiotic chemical reactivity methods as replacements for animal (in vivo) assays”. In this paper, we broaden the definition to include not only experimental methods, but also the possibility of capturing the information through a variety of computational approaches. In addition, the pur- pose of this paper is not to assess the use of in chemico approaches as a replacement (although this of course is desirable), but as a source of infor- mation, in combination with many others, on which to base a conclusion regarding the hazard associated with a particular chemical. Relating mechanistic organic chemistry to toxicology The basis of the in chemico approach, and the attempt to capture the information through in silico means, is mechanistic organic chemistry. The appli- cation of the approach is through the translation of the mechanisms into toxicologically meaningful in silico chemistry. There has been a particular empha- sis of capturing the reactivity of electrophiles (which can react covalently with biological nucleophiles). However, the role of other reactive species in toxicol- ogy, e.g. nucleophiles, reactive oxygen species, free radicals etc., should not be overlooked and is amenable to in chemico modelling. The In Chemico–In Silico Interface: Challenges for Integrating Experimental and Computational Chemistry to Identify Toxicity Mark T.D. Cronin, Fania Bajot, Steven J. Enoch, Judith C. Madden, David W. Roberts and Johannes Schwöbel School of Pharmacy and Chemistry, Liverpool John Moores University, UK Summary — A number of toxic effects are brought about by the covalent interaction between the toxicant and biological macromolecules. In chemico assays are available that attempt to identify reactive com- pounds. These approaches have been developed independently for pharmaceuticals and for other non- pharmaceutical compounds. The assays vary widely in terms of the macromolecule (typically a peptide) and the analytical technique utilised. For both sets of methods, there are great opportunities to capture in chemico information by using in silico methods to provide computational tools for screening purposes. In order to use these in chemico and in silico methods, integrated testing strategies are required for individ- ual toxicity endpoints. The potential for the use of these approaches is described, and a number of rec- ommendations to improve this extremely useful technique, in terms of implementing the Three Rs in toxicity testing, are presented. Key words: alternatives, in chemico, in silico, integrated testing strategy, reactivity, toxicity. Address for correspondence: Mark Cronin, School of Pharmacy and Chemistry, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF, UK. E-mail: m.t.cronin@ljmu.ac.uk ATLA 37, 513–521, 2009 513