DDI Induction

Cytochrome P450 induction forms part of in vitro experimental ADME services. A new test compound’s induction profile should routinely be evaluated.

These studies include but are not limited to:

  • CYP1A2, CYP2B6 and CYP3A4

  • Other Phase I enzymes including monoamine oxidase (MAO), flavin monooxygenase (FMO), xanthine oxidase (XO), and alcohol/aldehyde dehydrogenase

  • Phase II enzymes including UDP glucuronosyl transferases (UGTs)

Human hepatocytes continue to serve as the system of choice for evaluating enzyme induction in vitro, both with freshly isolated human hepatocytes and cryopreserved hepatocytes available for routine use.

When determining the enzyme induction potential of new test compounds using cultured human hepatocytes or HepaRG™ cells, the following elements must be considered:

  • Inter-individual variability
  • Changes in mRNA level of target genes as an endpoint (activity level is also evaluated at Eurosafe)
  • The inclusion of vehicle controls, positive controls (usually known strong inducers), and negative controls (usually known non-inducers) in the experiment.

Initially CYP1A2, CYP2B6, and CYP3A induction is evaluated (FDA guidance on DDI studies).

If no induction of CYP3A4/5 enzymes is observed, it is unnecessary to evaluate the induction potential of CYP2C enzymes because both CYP3A4/5 and CYP2C enzymes are induced via activation of pregnane X nuclear receptor (PXR).

If CYP3A4/5 induction does occur, however, it is then necessary to evaluate the potential of CYP2C induction.

DDI induction

We examine fold-change in CYP enzyme mRNA levels when incubated with the test compound by using cutoffs as described in FDA guidelines.

Eurosafe also evaluates of drug binding to microsomal proteins and cells in order to evaluate free fraction (fumic, fuhep) and also to predict hepatic clearance or drug-drug interaction potential in vivo, utilizing in vitro microsomal metabolic data.

DDI Inhibition

Inhibition of metabolism enzymes (CYP and UGT) by a new test compound may decrease the metabolism of co-medicated drugs. The potential of this test compound to inhibit enzymes is usually investigated to elucidate the precise inhibition mechanisms (reversible or time-dependent) as well as inhibition potency (e.g., Ki). FDA guidelines.

Liver microsomes are recommended for screenings and for mechanistic evaluations. Alternatively for systematic approaches, human hepatocytes or HepaRG™ cells can be used to determine the CYP inhibition potential of test compounds in the cellular environment.

Screening assays are performed with up to 9 cytochrome P450 enzymes (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4/5) using 10 probe reactions. The Direct Inhibition assay delivers IC50 values towards each CYP enzyme. The Time Dependent Inhibition assay yields direct and shifted IC50 values for each CYP enzyme.

Following IC50 determination, we determine Ki for the test compound against the appropriate enzyme. This parameter elucidates the potency of inhibition as well as the type of inhibition (i.e. competitive, non-competitive, uncompetitive or mixed). Such information can be used to estimate the impact of any potential in vivo interactions.

Eurosafe also evaluates of drug binding to microsomal proteins and cells in order to evaluate free fraction (fumic, fuhep) and also to predict hepatic clearance or drug-drug interaction potential in vivo, utilizing in vitro microsomal metabolic data.

DDI Phenotyping

In order to predict DDI, it is crucial to understand which enzymes (Phase I and phase II) are responsible for the metabolism of a given test compound.

There are three well-characterized methods recognized by the FDA for identifying the individual CYP enzymes involved in a drug’s metabolism.

- Method 1 uses a bank of human liver microsomes characterized for CYP activity


- Method 2 uses individual human recombinant CYP enzymes


- Method 3 uses specific chemicals or antibodies as specific enzyme inhibitors



- Method 3 bis: Eurosafe proposes the use of Silensomes™.

Silensomes™ are validated human-pooled liver microsomes (HLMs) which are chemically and irreversibly inactivated for a specific CYP450 using mechanism based inhibitors (MBI). This approach allows quantitative phenotyping by determining metabolic contribution (fm) instead of RAF evaluation.

It is recommended that at least two of the three methods above be performed in order to identify the specific enzyme(s) responsible for a given drug’s metabolism.


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Last posters

  • In-silico modelization of compounds interaction with Bile salt Export Pump (BSEP): an alternative approach to predict hepatotoxicity


    BSEP is an efflux transporter protein present in the hepatocytes membrane that plays important role in bile acid flow from hepatocyte cell into the bile canaliculi (1,2). Impaired BSEP activity due to drug interaction leads to accumulation of bile acid within the hepatocyte cells and results in cholestasis liver injury (DILI)(3). However, due to the lack of a X-ray structure of BSEP, there is no detail information about interaction of compounds with BSEP.

  • Cosmetics exposure data - Particular case of children from birth


    It is commonly accepted that the Skin Surface Area over Body weight ratio (SA/BW) of infants and children is higher as compared with that in adults. Regarding the safety assessment process of cosmetic ingredients, Toxicological Reference Values (TRV) are expressed as mg/kg bw and the change in the ratio of surface area/body weight compared with an adult would give, according to A.G. Renwick (1998), a discrepancy of 2.3-fold for children at birth (except premature infants). Such inter individual variation is already covered by the generally accepted default value of 100 calculated for individual ingredients which is composed of 10 for interspecies variations (4.0 for toxicokinetics) and 10 for human variability, covering toxicokinetic (3.2) and toxicodynamic (3.2) differences between children and adults (A.G. Renwick (1998) and SCCS (2018)). Scheuplein et al. (2002) estimate that an uncertainty factor of 10 applied to this intra-species variability is a sufficiently powerful parameter to take into account child/adult variability, for children over 6 months old. This estimate may not be relevant for children under 6 months of age, in the absence of developmental or systemic toxicity studies. Given the ambiguity of these data, the question was raised whether the use of an increased uncertainty/safety factor would be relevant to cover children at birth exposure to cosmetic ingredients and ensure their safety under normal and reasonably foreseeable conditions of use.

  • In silico prediction of Structure and compound affinity of Multidrug Resistance-associated Protein 2 (MRP2) for predicting hepatotoxicity.


    MRP2 is an unidirectional efflux transporter mainly present in liver, that primarily transports organic anions, including drug conjugates and conjugated bilirubin. MRP2 supports a function in the terminal excretion and detoxification of endogenous and xenobiotic organic anions. Due to the lack of a X-ray structure of MRP2, there is no detail information about interaction of compounds with MRP2.

  • Safety assessment of cosmetic ingredients for skin sensitization using QSAR in silico tool


    Skin sensitization methods are developed to protect workers and consumers from chemical exposures. In 2012, OECD Test Guideline 168 (TG) addressed the 5 Key Events (KE) of the skin sensitization Adverse Outcome Pathway (AOP)[1]. To respond to cellular and tissue events(KE2, KE3) in vitro alternative methods were well described such as OECD TG 442C (Direct Peptide Reactivity Assay, DPRA); OECD TG 442D (KeratinoSens™) and OECD TG 442E (human Cell Line Activation Test, h-CLAT). The two biomarkers based test SENS-IS and the Genomic Allergic Detection Test (GARD) are under consideration by the OECD for the development of the respective TGs.

  • Essential oils mixtures safety-in-use evaluation in personal care products


    Essentials oil are defined as complex natural substances. Their composition varies according to the geographical origin of the plant, the part of the plant used, the conditions of production or the method of production. The number of components present in these volatile oils can be very high, despite a structural variation rather limited (3 major atoms: carbon, hydrogen and oxygen). All these characteristics justify the difficulty of carrying out a reliable and relevant toxicological assessment of these ingredients, which are very popular in cosmetic industry. Thanks to chromatographic methods, it is now possible to know the nature and concentration of all the compounds present in an essential oils mixture. Thus, trhough analysis reports obtained by chromatography, the maximum acceptable concentration of a mixture for a given finished cosmetic product can be determined on the basis of the compound identified as limiting. The toxicological approach here applied is mainly based on the QRA methodology (*Qualitative Risk Assessment*) used for the calculation of IFRA certificates. Each compound identified by chromatography has a toxicity reference value (TRV) and a theoretical skin absorption rate. In the absence of TRV, the Threshold of Toxicological Concern approach (TTC - Cramer) will be applied.