Chemical Safety Assessment Using Read-Across: Assessing the Use of Novel Testing Methods to Strengthen the Evidence Base for Decision Making

Background: Safety assessment for repeated dose toxicity is one of the largest challenges in the process to replace animal testing. This is also one of the proof of concept ambitions of SEURAT-1, the largest ever European Union research initiative on alternative testing, co-funded by the European Commission and Cosmetics Europe. This review is based on the discussion and outcome of a workshop organized on initiative of the SEURAT-1 consortium joined by a group of international experts with complementary knowledge to further develop traditional read-across and include new approach data. Objectives: The aim of the suggested strategy for chemical read-across is to show how a traditional read-across based on structural similarities between source and target substance can be strengthened with additional evidence from new approach data—for example, information from in vitro molecular screening, “-omics” assays and computational models—to reach regulatory acceptance. Methods: We identified four read-across scenarios that cover typical human health assessment situations. For each such decision context, we suggested several chemical groups as examples to prove when read-across between group members is possible, considering both chemical and biological similarities. Conclusions: We agreed to carry out the complete read-across exercise for at least one chemical category per read-across scenario in the context of SEURAT-1, and the results of this exercise will be completed and presented by the end of the research initiative in December 2015

A liquid chromatography-tandem mass spectrometry method for the determination of 5-fluorouracil degradation rate by intact peripheral blood mononuclear cells

5-Fluorouracil (5-FU) is a major chemotherapy drug used for the treatment of tumors. It is catabolized mainly by dihydropyrimidine dehydrogenase, and patients with a complete or partial deficiency of dihydropyrimidine dehydrogenase activity are at risk of developing severe 5-FU-associated toxicity. The aim of this study was to demonstrate that intact peripheral blood mononuclear cells (PBMCs) can be an effective model to evaluate the degradation rate of 5-FU. We developed a sensitive and specific liquid chromatography-tandem mass spectrometry method to measure in vitro the rate of 5-FU degradation by intact PBMC. 5-FU degradation rate was determined by measuring the decrease of a fixed amount of 5-FU (10 μg/mL) added to a solution of PBMC, after 2 hours incubation, expressed as nanogram per milliliter of 5-FU degraded per minute × 10 cells. Freshly prepared intact PBMC can degrade efficiently in vitro-added 5-FU. The assay consists of 3 steps: (1) PBMC isolation from peripheral blood, (2) PBMC incubation with 5-FU in vitro for different times, and (3) determination of 5-FU amount to calculate the degradation rate. 5-FU was analyzed by a Q Trap 2000 triple quadrupole/ion trap mass spectrometer in the multiple-reaction-monitoring modes. The chromatographic separation was accomplished using a C18 column with a run time of 16 minutes. By analyzing samples from 39 patients with no 5-FU toxicity, the mean 5-FU degradation rate was 1.85 ± 0.50 ng•mL•min × 10 cells. The assessment of a test to measure 5-FU degradation rate in PBMC of patients before 5-FU administration could represent a prescreening method for evaluating the possible toxicity of this drug as an aid to set up a personalized medicine approach for each patient. Copyright © 2009 by Lippincott Williams & Wilkins

Serum levels of risperidone and its metabolite, 9-hydroxyrisperidone: Correlation between drug concentration and clinical response

The aim of this study was to assess a method able to analyze serum levels of risperidone (RIS) and its metabolite, 9-hydroxyrisperidone (9-OH-RIS), and to investigate possible relationships between changes in serum concentrations of these drugs and clinical measures, so to identify early markers of treatment response. The authors developed a sensitive and specific liquid chromatography-tandem mass spectrometry method to measure RIS and its metabolite in serum. Fifteen RIS-naive patients were admitted to an acute psychiatric care unit and treated with 4-6 mg/d oral RIS. At days 7 and 21 of hospital stay, serum levels were measured; clinical scales and serum prolactin were assessed. RIS and its metabolite were analyzed by a Q-Trap 2000 triple quadrupole/ion trap mass spectrometer in the multiple reaction-monitoring mode. Chromatographic separation was accomplished using a cyano column with an analytical run of 9 minutes. The calibration curve exhibited consistent linearity and reproducibility in the range 0-100 ng/mL for both analytes. Lower limit of quantification was 0.2 ng/mL; limit of detection, for a signal to noise ratio of 3, was 0.05 ng/mL for both analytes. Serum RIS and 9-OH-RIS levels increased at day 7, reaching a steady state, and remaining constant up to day 21. Scores on psychopathology rating scales decreased; serum prolactin and neurological rating scale for extrapyramidal effects rose at day 7 and remained stable thereafter. No correlation was found between serum concentration values, including sum and ratio of RIS and 9-OH-RIS, and any of the other clinical values (serum prolactin and clinical scales). These data indicate that clinical changes are related to the achievement of steady state levels of RIS and its metabolite and are maintained, but not continued, with continued RIS treatment. Therapeutic drug monitoring of RIS and its metabolites is not recommended as a routine procedure in patients with psychotic disorders. Copyright © 2009 by Lippincott Williams & Wilkins

Establishing a systematic framework to characterise in vitro methods for human hepatic metabolic clearance

Abstract Hepatic metabolic clearance is one of the most important factors driving the overall kinetics of chemicals including substances used in various product categories such as pesticides, biocides, pharmaceuticals, and cosmetics. A large number of in vitro systems from purified isozymes and subcellular organelles to hepatocytes in simple cultures and in complex scaffold setups are available for measuring hepatic metabolic clearance for different applications. However, there is currently no approach for systematically characterising and comparing these in vitro methods in terms of their design, applicability and performance. To address this, existing knowledge in the field of in vitro human hepatic metabolic clearance methods was gathered and analysed in order to establish a framework to systematically characterise methods based on a set of relevant components. An analogous framework would be also applicable for non-human in vitro systems. The components are associated with the biological test systems used (e.g. subcellular or cells), the in vitro method (e.g. number of cells, test item solubility), related analytical techniques, data interpretation methods (based on substrate depletion/metabolite formation), and performance assessments (precision and accuracy of clearance measurements). To facilitate the regulatory acceptance of this class of methods, it is intended that the framework provide the basis of harmonisation work within the OECD