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Improved analytical method for monitoring exposure to volatile compounds for occupational risk prevention
Abstracts / Toxicology Letters 189S (2009) S57–S273
P14 Comparative evaluation of (Q)SAR models for predicting genotoxicity endpoints Mandy Osterloh-Quiroz 1,∗ , Lynn Kan 2 , Kathryn M. Kleinert 2 , Darrell R. Boverhof 2 1
Dow Europe GmbH, Toxicology and Environmental Research & Consulting, Horgen, Switzerland, 2 The Dow Chemical Company, Toxicology and Environmental Research & Consulting, Midland, MI, United States New chemical regulations such as REACH are advocating the use of non-testing approaches to fill data gaps. The knowledge-based expert system DEREK, the rule-based statistical system TOPKAT and the hybrid expert system OASIS (TIMES) are among the QSAR models recommended in the ECHA Guidance for REACH. However, the guidance does not provide an evaluation on the performance of these models. A comparative analysis of currently available QSAR models against experimental data is needed to gain experience surrounding the application of modeling approaches predicting chemical-induced toxicity. The Dow Chemical Company has generated extensive data on the genotoxic potential of a wide variety of chemical substances using the Ames test, HPRT test (mutagenicity) and the in vitro chromosome aberration assay (clastogenicity). In the scope of this project, 40 chemicals were analyzed with the models DEREK, TOPKAT and OASIS followed by an evaluation of the model predictions in comparison with experimental test data. For the mutagenicity endpoint, the overall concordance between experimental data and predicted values was 72%, 69% and 72% with a specificity of 76%, 72% and 79% and a sensitivity of 57%, 57% and 43% for the models OASIS, TOPKAT and DEREK, respectively. For the clastogenicity endpoint, the overall concordance between experimental data and predicted values was 67% and 75% with a specificity of 79% and 89% and a sensitivity of 20% and 20% for the models OASIS and DEREK, respectively. Overall the performance of the three models was comparable, showing a high specificity but rather low sensitivity. doi:10.1016/j.toxlet.2009.06.383
P15 Cardiac ion channel safety screening with the CytoPatchTM instrument Olaf Scheel ∗ , Gesa Rascher-Eggstein ∗ , Thomas Knott
S261
shown for patch clamp measurement on the cardiac Kv1.5 ion channel. doi:10.1016/j.toxlet.2009.06.384
P16 Evaluation of inflammatory, cytotoxic, genotoxic BAL parameters and histological findings after exposure to amosite, refractory ceramic fibres and cigarette smoke Marta Hurbankova 1,∗ , Silvia Cerna 1 , Milan Beno 1 , Magdalena Barancokova 1 , Martina Valachovicova 1 , Sona Wimmerova 1 , Zuzana Kovacikova 1 , Elisabeth Tatrai 2 1
Slovak Medical University, Respiratory Toxicology, Bratislava, Slovakia, 2 NIOH, Budapest, Hungary Refractory ceramic fibres (RCF) are used as one kind of asbestos substitutes. Because RCF are relatively durable and some RCF are respirable, they may present a potential health hazard by inhalation. The aim of study was: (1) to find and compare the effects of subchronic exposure to amosite (AMO), refractory ceramic fibres (RCF), cigarette smoke (CS) and combined exposure to AMO + CS and RCF + CS by inflammatory, cytotoxic and genotoxic parameters of bronchoalveolar lavage (BAL) and by lung histology, (2) to find out if smoking amplifies the possible adverse effect of RCF as it is known after combined exposure to asbestos + CS. After 6-month exposure the animals were exsanguinated and BAL was performed. Following BAL parameters were examined: BAL cell count; alveolar macrophages (AM) count, differential cell count (% of AM, polymorphonuclears and lymphocytes), % of immature AM, binucleated cells, viability and phagocytic activity of AM, frequency of micronuclei (MN), comet assay and histology of lung tissue. The results of our work suggest: (1) there are no great differences between AMO and RCF exposure and their combined effects with cigarette smoke in BAL inflammatory and cytotoxic parameters under our experimental conditions in BAL parameters; (2) RCF did not treated MN; comet assay results were not significant influenced after AMO or RCF exposure. (3) According to histological findings there are differences of lung tissue injury (fibrosis) between AMO (grade 8) and RCF (grade 5) exposure and there is no fibrosis effect (grade 0) after cigarette smoke exposure. Acknowledgements: This project has been supported by the grant APVT-21-011164 and MZ SR 2005/29-SZU-07. doi:10.1016/j.toxlet.2009.06.385
Cytocentrics AG, Screening Department, Rostock, Germany Drug induced QT-prolongation and cardiac arrhythmias are often the result of blocking the human ether-a-go-go related gene (hERG) K+ channel as an unwanted side effect. Screening against the hERG channel is therefore an important issue in pharmacology safety. Manual patch clamp is the gold standard for investigating ion channel modulation. The high data quality is reached at the expense of very low throughput and the need of an experienced operator. Here it is shown that with the CytoPatchTM instrument the high data quality known from manual patch clamp can be combined with a complete process and assay automation. This results in an increased throughput needed for screening purposes. The electrophysiological properties of the hERG ion channel and concentration response relationship of know hERG inhibiting compounds are determined with the CytoPatchTM instrument and are well comparable to data obtained with manual patch clamp. This is also
P17 Improved analytical method for monitoring exposure to volatile compounds for occupational risk prevention Carmen Estevan Martínez ∗ , Miguel Ángel Sogorb Sánchez, Eugenio Vilanova Gisbert University Miguel Hernández de Elche, Instituto de Bioingeniería, Elche, Spain The shoemaking and other leatherwear industries employ adhesives and other kinds of products containing volatile organic compounds. These substances are known to cause neurological disorders (Filley et al., 2004; Pastore et al., 1994). For adequate risk management purposes, it is necessary to determine the occupational exposure to volatile organic compounds. There are several
S262
Abstracts / Toxicology Letters 189S (2009) S57–S273
official validated methods, used for the characterization of occupational environments containing mixtures of volatile organic compounds, but none for the simultaneous determination of all the substances that are usually present in the leatherwear industry environment, like hexane or heptane isomers. Thus, an optimization and simplification of sample extraction and chromatographic parameters was carried out in this work to obtain a method capable of a fast and efficient determination of the most frequently found volatile compounds in this industry. The methodology is able to simultaneously detect 13 organic volatile compounds (nhexane and isomers, n-heptane and isomers, cyclohexane, toluene, benzene, ketones, trichloroethylene and ethyl acetate) in 11 min. Detection and quantification limits were ranging between 0.1 and 21.6 g/mL and 0.4 and 54.3 g/mL, respectively, which for an active captation sampling about 10 L of air; it represents less than 1% of the threshold limit value proposed by the EU and Spanish guidelines. The method has proved suitable for monitoring occupational exposure to these compounds in industries and it satisfies the simplicity, speed, sensitivity and efficacy conditions for the determination of volatile organic compounds in activated charcoal tubes. References Filley, C.M., Halliday, W., Kleinschmidt-DeMasters, B.K., 2004. The effects of toluene on the central nervous system. J. Neuropathol. Exp. Neurol. 63 (1), 1–12. Pastore, C., Marhuenda, D., Marti, J., Cardona, A., 1994. Early diagnosis of n-hexanecaused neuropathy. Muscle Nerve 17 (9), 981–986.
doi:10.1016/j.toxlet.2009.06.386
P18 Mitochondrial effects of chloramphenicol and linezolid in HepG2 cells Doris Höschele ∗ , Martina Wiertz Bundesinstitut für Arzneimittel und Medizinprodukte, Bonn, Germany Several classes of antibiotics induce mitochondrial toxicity due to inhibition of mitochondrial protein synthesis, including chloramphenicol and linezolid. Clinically, inhibition of mitochondrial protein synthesis may lead to severe adverse effects in patients treated with chloramphenicol or linezolid. Inhibition of mitochondrial protein synthesis has been well examined in several published studies for these compounds. In this study, we investigated the effects of chloramphenicol and linezolid on mitochondrial DNA (mtDNA) content, expression of mtDNA encoded proteins of respiratory chain complexes and cellular lactate production in HepG2 cells. HepG2 cells were treated over 14 days with chloramphenicol (1, 10, or 100 M) or with linezolid (0.10 or 0.25 mg/mL). MtDNA gene expression and mtDNA content were analysed by real-time PCR. Mitochondrial function was determined by measuring cellular lactate production. Both compounds induced a time- and concentration-dependent increase in cellular lactate production, suggesting impairment of mitochondrial function. For chloramphenicol a concentrationdependent increase in mtDNA content was observed, whereas expression of mtDNA encoded proteins decreased, except for ND4L and ND1 genes which increased. No alteration in mtDNA content occurred after linezolid treatment. However, a concentrationdependent increase in gene expression of all mtDNA encoded proteins was observed.
This study shows that although chloramphenicol and linezolid are potent inhibitors of mitochondrial protein synthesis and induce mitochondrial dysfunction in HepG2 cells, there are some differences in mitochondrial effects between both compounds. This suggests that a universal mechanism for the mitochondrial toxicity of chloramphenicol and linezolid may not exist. doi:10.1016/j.toxlet.2009.06.387
P19 A computer tool for the assessment of potential combined acute effects of chemicals in mixtures Jonathan Côté 1,∗ , Franc¸ois Lemay 2 , Claude Viau 1 , Adolf Vyskocil 1 1
University of Montreal, Santé environnementale et santé au Travail, Montreal, Canada, 2 Institut de Recherche Robert-Sauvé en Santé et Sécurité au Travail, Montréal, Canada When chemicals agents are spilled in the environment because of accidents occurring during hazardous materials transport or in chemical industries, employees, first-aid workers, firefighters or the general population can be exposed for short periods to high concentrations of multiple chemicals agents. In those cases of acute exposures, the overall toxicity of a mixture may be greater than that associated with each of its individual compounds. In occupational health, consideration of this phenomenon has resulted in specific recommendations by the American Conference of Governmental Industrial Hygienists and in Quebec’s Regulation respecting occupational health and safety. These recommendations or prescriptions call for verification of possible addition of effects on a given organ of the human body caused by the concomitant inhalation of two or more hazardous substances in the air. This project was undertaken with the objective of developing a user-friendly computer tool to identify the possible additive acute effects of mixtures. The first step was to create a toxicological database of target organs and acute toxic effects on these organs for 200 hazardous substances. With this database, a computer tool was developed to calculate an acute mixture exposure index based on the Acute Exposure Guideline Levels (AEGL) developed by EPA. These values were established for 5 exposure durations (10 min, 30 min, 1 h, 4 h and 8 h) and 3 toxicity levels (AEGL-1, AEGL-2 and AEGL-3). With this information, the computer tool estimates the possible addition of acute effects based on exposure concentrations of individual substances provided by the user. doi:10.1016/j.toxlet.2009.06.388
P20 The impact of drug monitoring in a case of drug resistant neonate arrhythmia Maria Toutoudaki 1,∗ , Ioannis Germanakis 2 , George Briassoulis 3 , Manolis Tzatzarakis 1 , Maria Christakis-Hampsas 1 , Aristidis Tsatsakis 1 1
University of Crete, Lab of Toxicology, Medical School, Heraklion, Greece, 2 University Hospital, Pediatric & Fetal Cardiology Unit, Dpt of Pediatrics, Heraklion, Greece, 3 University Hospital, Childrens Intensive Care Unit/Dept. of Pediatrics, Heraklion, Greece Purpose: A case of refractory neonatal supraventricular tachycardia, necessitating multidrug treatment including propafenone and close drug level monitoring is described.
P14 Comparative evaluation of (Q)SAR models for predicting genotoxicity endpoints Mandy Osterloh-Quiroz 1,∗ , Lynn Kan 2 , Kathryn M. Kleinert 2 , Darrell R. Boverhof 2 1
Dow Europe GmbH, Toxicology and Environmental Research & Consulting, Horgen, Switzerland, 2 The Dow Chemical Company, Toxicology and Environmental Research & Consulting, Midland, MI, United States New chemical regulations such as REACH are advocating the use of non-testing approaches to fill data gaps. The knowledge-based expert system DEREK, the rule-based statistical system TOPKAT and the hybrid expert system OASIS (TIMES) are among the QSAR models recommended in the ECHA Guidance for REACH. However, the guidance does not provide an evaluation on the performance of these models. A comparative analysis of currently available QSAR models against experimental data is needed to gain experience surrounding the application of modeling approaches predicting chemical-induced toxicity. The Dow Chemical Company has generated extensive data on the genotoxic potential of a wide variety of chemical substances using the Ames test, HPRT test (mutagenicity) and the in vitro chromosome aberration assay (clastogenicity). In the scope of this project, 40 chemicals were analyzed with the models DEREK, TOPKAT and OASIS followed by an evaluation of the model predictions in comparison with experimental test data. For the mutagenicity endpoint, the overall concordance between experimental data and predicted values was 72%, 69% and 72% with a specificity of 76%, 72% and 79% and a sensitivity of 57%, 57% and 43% for the models OASIS, TOPKAT and DEREK, respectively. For the clastogenicity endpoint, the overall concordance between experimental data and predicted values was 67% and 75% with a specificity of 79% and 89% and a sensitivity of 20% and 20% for the models OASIS and DEREK, respectively. Overall the performance of the three models was comparable, showing a high specificity but rather low sensitivity. doi:10.1016/j.toxlet.2009.06.383
P15 Cardiac ion channel safety screening with the CytoPatchTM instrument Olaf Scheel ∗ , Gesa Rascher-Eggstein ∗ , Thomas Knott
S261
shown for patch clamp measurement on the cardiac Kv1.5 ion channel. doi:10.1016/j.toxlet.2009.06.384
P16 Evaluation of inflammatory, cytotoxic, genotoxic BAL parameters and histological findings after exposure to amosite, refractory ceramic fibres and cigarette smoke Marta Hurbankova 1,∗ , Silvia Cerna 1 , Milan Beno 1 , Magdalena Barancokova 1 , Martina Valachovicova 1 , Sona Wimmerova 1 , Zuzana Kovacikova 1 , Elisabeth Tatrai 2 1
Slovak Medical University, Respiratory Toxicology, Bratislava, Slovakia, 2 NIOH, Budapest, Hungary Refractory ceramic fibres (RCF) are used as one kind of asbestos substitutes. Because RCF are relatively durable and some RCF are respirable, they may present a potential health hazard by inhalation. The aim of study was: (1) to find and compare the effects of subchronic exposure to amosite (AMO), refractory ceramic fibres (RCF), cigarette smoke (CS) and combined exposure to AMO + CS and RCF + CS by inflammatory, cytotoxic and genotoxic parameters of bronchoalveolar lavage (BAL) and by lung histology, (2) to find out if smoking amplifies the possible adverse effect of RCF as it is known after combined exposure to asbestos + CS. After 6-month exposure the animals were exsanguinated and BAL was performed. Following BAL parameters were examined: BAL cell count; alveolar macrophages (AM) count, differential cell count (% of AM, polymorphonuclears and lymphocytes), % of immature AM, binucleated cells, viability and phagocytic activity of AM, frequency of micronuclei (MN), comet assay and histology of lung tissue. The results of our work suggest: (1) there are no great differences between AMO and RCF exposure and their combined effects with cigarette smoke in BAL inflammatory and cytotoxic parameters under our experimental conditions in BAL parameters; (2) RCF did not treated MN; comet assay results were not significant influenced after AMO or RCF exposure. (3) According to histological findings there are differences of lung tissue injury (fibrosis) between AMO (grade 8) and RCF (grade 5) exposure and there is no fibrosis effect (grade 0) after cigarette smoke exposure. Acknowledgements: This project has been supported by the grant APVT-21-011164 and MZ SR 2005/29-SZU-07. doi:10.1016/j.toxlet.2009.06.385
Cytocentrics AG, Screening Department, Rostock, Germany Drug induced QT-prolongation and cardiac arrhythmias are often the result of blocking the human ether-a-go-go related gene (hERG) K+ channel as an unwanted side effect. Screening against the hERG channel is therefore an important issue in pharmacology safety. Manual patch clamp is the gold standard for investigating ion channel modulation. The high data quality is reached at the expense of very low throughput and the need of an experienced operator. Here it is shown that with the CytoPatchTM instrument the high data quality known from manual patch clamp can be combined with a complete process and assay automation. This results in an increased throughput needed for screening purposes. The electrophysiological properties of the hERG ion channel and concentration response relationship of know hERG inhibiting compounds are determined with the CytoPatchTM instrument and are well comparable to data obtained with manual patch clamp. This is also
P17 Improved analytical method for monitoring exposure to volatile compounds for occupational risk prevention Carmen Estevan Martínez ∗ , Miguel Ángel Sogorb Sánchez, Eugenio Vilanova Gisbert University Miguel Hernández de Elche, Instituto de Bioingeniería, Elche, Spain The shoemaking and other leatherwear industries employ adhesives and other kinds of products containing volatile organic compounds. These substances are known to cause neurological disorders (Filley et al., 2004; Pastore et al., 1994). For adequate risk management purposes, it is necessary to determine the occupational exposure to volatile organic compounds. There are several
S262
Abstracts / Toxicology Letters 189S (2009) S57–S273
official validated methods, used for the characterization of occupational environments containing mixtures of volatile organic compounds, but none for the simultaneous determination of all the substances that are usually present in the leatherwear industry environment, like hexane or heptane isomers. Thus, an optimization and simplification of sample extraction and chromatographic parameters was carried out in this work to obtain a method capable of a fast and efficient determination of the most frequently found volatile compounds in this industry. The methodology is able to simultaneously detect 13 organic volatile compounds (nhexane and isomers, n-heptane and isomers, cyclohexane, toluene, benzene, ketones, trichloroethylene and ethyl acetate) in 11 min. Detection and quantification limits were ranging between 0.1 and 21.6 g/mL and 0.4 and 54.3 g/mL, respectively, which for an active captation sampling about 10 L of air; it represents less than 1% of the threshold limit value proposed by the EU and Spanish guidelines. The method has proved suitable for monitoring occupational exposure to these compounds in industries and it satisfies the simplicity, speed, sensitivity and efficacy conditions for the determination of volatile organic compounds in activated charcoal tubes. References Filley, C.M., Halliday, W., Kleinschmidt-DeMasters, B.K., 2004. The effects of toluene on the central nervous system. J. Neuropathol. Exp. Neurol. 63 (1), 1–12. Pastore, C., Marhuenda, D., Marti, J., Cardona, A., 1994. Early diagnosis of n-hexanecaused neuropathy. Muscle Nerve 17 (9), 981–986.
doi:10.1016/j.toxlet.2009.06.386
P18 Mitochondrial effects of chloramphenicol and linezolid in HepG2 cells Doris Höschele ∗ , Martina Wiertz Bundesinstitut für Arzneimittel und Medizinprodukte, Bonn, Germany Several classes of antibiotics induce mitochondrial toxicity due to inhibition of mitochondrial protein synthesis, including chloramphenicol and linezolid. Clinically, inhibition of mitochondrial protein synthesis may lead to severe adverse effects in patients treated with chloramphenicol or linezolid. Inhibition of mitochondrial protein synthesis has been well examined in several published studies for these compounds. In this study, we investigated the effects of chloramphenicol and linezolid on mitochondrial DNA (mtDNA) content, expression of mtDNA encoded proteins of respiratory chain complexes and cellular lactate production in HepG2 cells. HepG2 cells were treated over 14 days with chloramphenicol (1, 10, or 100 M) or with linezolid (0.10 or 0.25 mg/mL). MtDNA gene expression and mtDNA content were analysed by real-time PCR. Mitochondrial function was determined by measuring cellular lactate production. Both compounds induced a time- and concentration-dependent increase in cellular lactate production, suggesting impairment of mitochondrial function. For chloramphenicol a concentrationdependent increase in mtDNA content was observed, whereas expression of mtDNA encoded proteins decreased, except for ND4L and ND1 genes which increased. No alteration in mtDNA content occurred after linezolid treatment. However, a concentrationdependent increase in gene expression of all mtDNA encoded proteins was observed.
This study shows that although chloramphenicol and linezolid are potent inhibitors of mitochondrial protein synthesis and induce mitochondrial dysfunction in HepG2 cells, there are some differences in mitochondrial effects between both compounds. This suggests that a universal mechanism for the mitochondrial toxicity of chloramphenicol and linezolid may not exist. doi:10.1016/j.toxlet.2009.06.387
P19 A computer tool for the assessment of potential combined acute effects of chemicals in mixtures Jonathan Côté 1,∗ , Franc¸ois Lemay 2 , Claude Viau 1 , Adolf Vyskocil 1 1
University of Montreal, Santé environnementale et santé au Travail, Montreal, Canada, 2 Institut de Recherche Robert-Sauvé en Santé et Sécurité au Travail, Montréal, Canada When chemicals agents are spilled in the environment because of accidents occurring during hazardous materials transport or in chemical industries, employees, first-aid workers, firefighters or the general population can be exposed for short periods to high concentrations of multiple chemicals agents. In those cases of acute exposures, the overall toxicity of a mixture may be greater than that associated with each of its individual compounds. In occupational health, consideration of this phenomenon has resulted in specific recommendations by the American Conference of Governmental Industrial Hygienists and in Quebec’s Regulation respecting occupational health and safety. These recommendations or prescriptions call for verification of possible addition of effects on a given organ of the human body caused by the concomitant inhalation of two or more hazardous substances in the air. This project was undertaken with the objective of developing a user-friendly computer tool to identify the possible additive acute effects of mixtures. The first step was to create a toxicological database of target organs and acute toxic effects on these organs for 200 hazardous substances. With this database, a computer tool was developed to calculate an acute mixture exposure index based on the Acute Exposure Guideline Levels (AEGL) developed by EPA. These values were established for 5 exposure durations (10 min, 30 min, 1 h, 4 h and 8 h) and 3 toxicity levels (AEGL-1, AEGL-2 and AEGL-3). With this information, the computer tool estimates the possible addition of acute effects based on exposure concentrations of individual substances provided by the user. doi:10.1016/j.toxlet.2009.06.388
P20 The impact of drug monitoring in a case of drug resistant neonate arrhythmia Maria Toutoudaki 1,∗ , Ioannis Germanakis 2 , George Briassoulis 3 , Manolis Tzatzarakis 1 , Maria Christakis-Hampsas 1 , Aristidis Tsatsakis 1 1
University of Crete, Lab of Toxicology, Medical School, Heraklion, Greece, 2 University Hospital, Pediatric & Fetal Cardiology Unit, Dpt of Pediatrics, Heraklion, Greece, 3 University Hospital, Childrens Intensive Care Unit/Dept. of Pediatrics, Heraklion, Greece Purpose: A case of refractory neonatal supraventricular tachycardia, necessitating multidrug treatment including propafenone and close drug level monitoring is described.