Overcoming some challenges of the Local Lymph Node Assay (LLNA)

Abstracts / Toxicology Letters 211S (2012) S43–S216

recent exposure. Therefore, it is imperative the use of an alternative biological matrix that may provide relevant and important retrospective information about the use of drugs, namely cocaine and morphine which are commonly abused. Methods: A qualitative and quantitative method for the simultaneous determination of cocaine and morphine in human hair was developed and validated. After decontamination, hair samples (20 mg) were incubated with a mixture of methanol/hydrochloric acid (2:1) at 65 ◦ C overnight in order to extract the XBs of the matrix. Samples were cleaned-up by mixed-mode solid-phase extraction (SPE), XBs were derivatized with N-methyl-N-(trimethylsilyl) trifluoroacetamide and then analyzed by gas chromatography/electron impact/mass spectrometry (GC/EI/MS). Results of the study: The developed method proved to be specific, accurate and precise across the calibration range (0.25–10 ng/mg), where good linearity was observed for both the analytes with correlation coefficients ranging 0.999 and 0.9993. The coefficients of variation oscillated between 0.18% and 16.16%. The limits of quantification (LOQ) were 0.04 and 0.05 ng/mg for cocaine and morphine, respectively. The proposed GC/EI/MS method can be successfully applied in the screening and quantification of these XBs in real cases, namely in clinical and forensic toxicology. doi:10.1016/j.toxlet.2012.03.542

P23-07 Tramadol and O-desmethyltramadol quantification in hair samples by GC–MS Ricardo Dinis-Oliveira 1 , Sandra Pinho 1 , Ana Oliveira 2 , Félix Carvalho 2 , Roxana Falcão Moreira 1 ISCS-N/CESPU, Crl, Portugal, 2 Faculty of Pharmacy, University of Porto, Portugal

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Purpose: Abuse of tramadol and related fatal intoxications has been increasing. Hair analysis is the ideal matrix to evidence cumulative long-term exposure. Besides tramadol, it is important to quantify its main metabolite, O-desmethyltramadol (M1), since when present in hair represents internal exposure and it is much more active than tramadol itself. Until now, there is no validated technique to simultaneously quantify tramadol and M1 in hair. Methods: A gas-chromatography/mass spectrometry (GC–MS) method with solid-phase-extraction (SPE) for simultaneous determination of tramadol and M1 in human hair samples was developed and validated. Hair samples (60 mg), were subjected to decontamination with dichloromethane, water and acetone, and then extracted with methanol in ultrasonic bath. Samples were then cleaned-up with solid-phase extraction using mixed-mode MCX cartridges. Derivatization was performed using ethyl acetate and bis(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane (BSTFA + 1%TMCS) and then analytes were analyzed by GC–MS. Results of the study: Validation of the method was performed working with spiked hair samples. The method proved to be selective since there were no matrix interferences. The regression analysis for tramadol and M1 was linear in the range of 0.1–20 ng/mg with detection and quantification limits of 0.0003 and 0.0013 ng/mg for tramadol and 0.0001 and 0.0006 ng/mg for M1 respectively. The present method was further applied to six clinical cases of longterm tramadol administration. doi:10.1016/j.toxlet.2012.03.543

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P23-08 Characterization of crystalline deposits in rat kidney using NMR, LC/MS and MALDI MSI Benita Forngren 1 , Anna Nilsson 2 , Sivert Bjurström 3 , Håkan Andersson 3 , Anita Annas 3 , Dennis Hellgren 3 , Alexander Svanhagen 3 , Per Andren 2 , Johan Lindberg 3 AstraZeneca, Sweden, 2 Pharm. Biosciences, Uppsala University, Sweden, 3 Safety Assessment, AstraZeneca, Sweden

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Wistar rats administered with a microsomal prostaglandin E synthase 1 (mPGES-1) inhibitor for 1 week, showed severe kidney injury. Clinical pathology indicated renal damage (increased plasma levels of urea, creatinine and potassium) in the high dose rats. The histopathological examination of kidneys mainly showed marked tubular degeneration/regeneration, crystalline deposits in tubules surrounded by inflammatory reaction, dilated tubules with cellular remnants in lumen and presence of crystals in the pelvis. One hypothesis was that toxicity was caused by precipitation of the metabolite bisulphonamide which is formed by hydrolysis of the parent compound, as this metabolite is known to have poor solubility. The aim of this study was to determine the chemical identity of the crystal deposits, by a combination of analyses using nuclear magnetic resonance (NMR), liquid chromatography/mass spectrometry (LC/MS) and matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI). Analysis was performed by extraction of kidney tissue, manual dissection of precipitated crystals from kidney tissue sections and MALDI MSI of kidney sections. Rats administered with vehicle served as controls in the experiments. Analysis of crystals that were dissected directly from kidney sections and analysed with LC/MS and NMR, clearly showed that the crystals contained bisulfonamide in all investigated samples. In addition, MALDI MS imaging was used to correlate the spatial distribution of bisulfonamide (m/z 234.98) on kidney sections to histological defined areas. These combined results propose precipitation of bisulphonamide as a possible cause to the kidney toxicity observed in this study. doi:10.1016/j.toxlet.2012.03.544

P23-09 Overcoming some challenges of the Local Lymph Node Assay (LLNA) Katharina Lütkenhaus, Daniela Stelter, Achim Albrecht BSL Bioservice Scientific Laboratories, Germany The LLNA is a validated substitute for guinea pig tests for identifying potential skin sensitizer. By measuring the proliferation rate of the lymphocytes in the auricular lymphnodes, the LLNA provides quantitative data about the skin sensitizing potential of a substance. Although the LLNA is a robust assay and allows a reproducible characterization of the test substance, some challenges occur: (A) the OECD TG 429 only suggests organic solvents and organic-aqueous mixtures, but no aqueous vehicle for testing aqueous soluble test substances is generally approved. (B) As the disposal of 3H is due to its long half life (∼12.3 years) an environmental as well as a cost problem, an alternative non-radioactive approach (OECD TG 442B), was established. (A) We investigated that 2% carboxymethylcellulose in water is a suitable vehicle for hydrophilic substances which could be approved by the positive control substance alpha-

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Abstracts / Toxicology Letters 211S (2012) S43–S216

Hexylcinnamaldehyde in the conventional test protocol. (B) While in the conventional LLNA the incorporation of 3H-methyl thymidine is measured by beta-scintillation counting as disintegrations per minute, in the non-radioactive LLNA the incorporation of 5bromo-2-deoxyuridine is measured by ELISA. We established and validated the non-radioactive LLNA in our laboratory and approved the robustness as well as reproducibility of this test method. Although the OECD TG 429 does not recommend any aqueous vehicle, we could show for the conventional LLNA that the measurement of hydrophilic substances can be performed by choosing an appropriate vehicle. Furthermore, the non-radioactive protocol provided in the OECD TG 442B could be established and validated in our laboratory. doi:10.1016/j.toxlet.2012.03.545

P23-10 The Cell Screening Facility at Science for Life Laboratory – Stockholm Bo Lundgren, Ulf Martens, Maria Häggblad SciLifeLab Stockholms University, Sweden Science for Life Laboratory – Stockholm (SciLifeLab) is a new Swedish national resource centre dedicated to large scale research in molecular biosciences and medicine. The major funding for SciLifeLab comes from strategic government grants. SciLifeLab – Stockholm is a joint collaboration between three universities, The Royal Institute of Technology (KTH), Karolinska Institute (KI) and Stockholm University (SU). The Cell Screening Facility at SciLifeLab use advanced robotics and micro plate technology for high throughput screening with RNAi or small molecules libraries in cell lines. The use of micro plates and robotics technologies have been extensively used for long time in the pharmaceutical industry mainly for finding, identify and characterize new drug targets. The major objectives for the Cell Screen Facility are to provide this technology to the academic community. Main activities • RNAi high throughputscreens and validation to identify new proteins involved in biological processes • Small molecule screens (inhibition toxicity) • Assay development • Map functional genetic networks. • Preclinical and in vitro toxicology support Major equipment • • • • • •

Janus 3 arm robot with 96 and 384 head Envison plate reader Cell washer and Flexdrop Echo 550 liquid dispenser RNAi libraries Chemical libraries

SciLifeLab – Stockholm could be engage in projects at three different levels: service, Collaborative and internal SciLifeLab projects for more information see contact info Contact info: [email protected] or www.scilifelab.se. doi:10.1016/j.toxlet.2012.03.546

P23-11 High dose ␤-cyclodextrin exposure via continuous or intermittent IV infusion in rats Harmke van Vugt, Ankie Schoenmakers, Maud Wasserman, Bianca van Rozendaal, Harry Emmen NOTOX a WIL Research Company, Netherlands During nonclinical drug safety testing, excipients are often needed to reach sufficiently high dose levels to achieve the required safety margin. ␤-Cyclodextrins are widely used to solubilize drugs, including those intended for intravenous (IV) administration. However, the possible effects of high doses of ␤-cyclodextrins in a nonclinical setting have not yet been fully characterized. In addition, higher doses may be tolerated when given via intermittent infusion. The effects of a high dose ␤-cyclodextrin, given via either continuous or intermittent IV infusion for 7 days, were studied in rats. Male Sprague-Dawley rats were catheterized via the femoral vein into the vena cava, the catheter was tunneled subcutaneously to the neck and externally attached to a harness and tether. 50% ␤-cyclodextrin was administered for 24-h (continuous) or 4-h (intermittent) by IV infusion at a dosing speed of 1.25 or 7.5 mL/kg/h, respectively, both resulting in a dose level of 15,000 mg/kg/day. The intermittent group received saline during the remaining 20 h/day at 0.4 mL/h. Control animals received 24-h continuous IV infusion with saline at 0.4 mL/h. No significant differences were observed between the two dosing scenarios. ␤-Cyclodextrin-related findings included reduced food intake, body weight loss and general poor condition. Main target organs were liver and kidneys, as reflected by changes in hematology, biochemistry and urinalysis parameters, macroscopic pathology, organ weights and microscopic pathology. At both continuous and intermittent IV infusion, the high level of 15,000 mg/kg/day exceeded the tolerated dose at which ␤cyclodextrin can be used as excipient in rats. doi:10.1016/j.toxlet.2012.03.547

P23-12 Spontaneous mutation frequency in normal human mammary gland tissue Katja Schmalbach, Leane Lehmann University of Wuerzburg, Germany Annually, over 57,000 women develop breast cancer in Germany. In particular mutations in tumor suppressor genes, e.g. p53, seem to play an important role in developing cancer. Up to now, lack of a method sensitive enough to determine the expected very low spontaneous mutation frequency (SMF) in normal mammary gland tissue precluded the investigation of the role of spontaneous mutations acquired in the p53 gene in epidemiological studies. The only test with the potential to determine low SMFs was the Random Mutation Capture (RMC) assay, a genotype selective method. Therefore, the suitability of the RMC assay to determine SMF in p53 gene in normal human mammary gland tissue was evaluated. The RMC assay was optimized concerning (i) DNA isolation, (ii) PCR conditions, and (iii) amount of mammary gland tissue. (i) Genomic DNA from normal human mammary gland tissue, obtained from healthy women who underwent mamma reduction surgery, was isolated using an extended proteinase k digestion prior to chloroform extraction. (ii) The target sequence