COMPARISON OF SELECTED IN VITRO ASSAYS FOR ASSESSING THE TOXICITY OF CHEMICALS AND THEIR MIXTURES Rola Azzi A thesis submitted for the degree of Doctor of Philosophy Chemical Safety and Applied Toxicology Laboratories School of Safety Science Faculty of Science The University of New South Wales June 2006 Certificate of Originality Certificate of Originality I hereby declare that this submission is my own work and that, to the best of my knowledge it contains no materials previously published or written by another person, nor material which to a substantial extent has been accepted for the award of any degree at UNSW or any other education institution, except where due acknowledgment is made in the thesis. Any contributions made to the research by others, which whom I have worked, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project’s design and conception or in style, presentation and linguistic expression is acknowledged. Rola Azzi June 2006 i Acknowledgments Acknowledgments I would like to express my sincerest gratitude to my supervisor Dr Amanda Hayes for her assistance, constant encouragement and expertise. Without her support and guidance this project would not have been possible. I am also grateful to Associate Professor Chris Winder, for his constant support, advice, and scientific expertise throughout my work on this project. I would also like to express my sincerest gratitude and appreciation to my uncle, Associate Professor Rachad Saliba, who was a constant support during the writing of this thesis, and was devoted to helping me grasp the science of statistics, and its ability to transform numbers into a story. To my colleagues at the School of Safety Science: Dr Christian Khalil, Mrs Fatma Lestari, Mrs Shahnaz Bakand and Mrs Aparna Manglik. I thank you for your friendship and support. To the special friends who shared this journey with me, you have always been there for me, even from across the seas. Thank you for the constant support and friendship, it made life much more meaningful. Special thanks go to my family who have always been there to support and encourage me in all my endeavours. A special thank you to my grandparents for believing in me so much. Last and most importantly to my parents and sister: you are my backbone in life. Thank you for the considerable sacrifices and supporting me in following my path in life even when it meant living in another country. I could not have made it through this thesis without your unconditional love and unlimited support in every possible way. This research is dedicated to my brother Tony. You are and will always be my inner strength, and my hope. I miss you. ii ‘Indulge your passion for science but let your science be human, and such as may have a direct reference to action and society’ (David Hume 1711-1776) (Enquiry Concerning Human Understanding: Introduction, 9) iii Abstract Abstract From a regulatory point of view, the main objective of acute toxicity testing is to classify chemicals according to their intrinsic toxicity. This is conventionally conducted on the basis of the animal LD50 methods however, this test has been widely criticised. Moreover, conventional toxicity testing focuses on single chemicals and often human exposures are to more than one chemical. This research study had two main objectives. The first was to investigate the accuracy of selected in vitro tests for predicting the acute toxic action of chemicals in rodents and humans, and in predicting the Globally Harmonised System of Classification and Labelling of Chemicals (GHS) categories. The second was to explore the relevance of in vitro tests in determining the nature of toxicological interactions (i.e. additive, antagonistic and synergistic) among binary and ternary chemical mixtures. A battery of cytotoxicity tests (MTS, NRU, LDH and ATP) assays were used to determine the toxicity of 21 chemicals spread across the GHS categories where mercuric chloride (GHS category 1) was the most toxic chemical and glycerol (GHS unclassified) the least toxic. Significant differences for the majority of test chemicals were found among all assays, highlighting the need for a battery of in vitro tests measuring different endpoints. The NRU assay was found to be a more sensitive measure of toxicity for most chemicals and predicted all of the GHS categories. In general, in vitro IC50 values correlated well with in vivo rodent (LD50); human acute toxicity (LDL0 and LC) data and published in vitro data. In addition, in vitro NOEC values correlated well with published TLV. Selected assays (MTS, NRU) were applied to binary (15) and ternary (5) chemical mixtures. Mixtures were prepared at mixture ratios proportional to the potency of individual components. Experimental data was used to assess the predictive capabilities of two approaches (mathematical model and concentration addition) commonly used by regulatory agencies which assume additive effect. However, all three interactions (antagonism, synergism and additivity) where observed in this study. These results suggest that these interactions cannot be excluded from toxicological risk assessments. The methods developed and information obtained from this study provide a comprehensive comparison between selected in vitro assays for assessing the toxicity of chemicals and their mixtures. iv Table of Contents Table of Contents Certificate0H of Originality………………………………………………………………..i286H Acknowledgments………………………………………………………………………1H ii287H Table2H of Contents……………………………………………………………………… v28H List3H of Tables …………………………………………………………………………..ix289H Listof4H Figures…………………………………………………………………………..xi290H Listof5H Abbreviations………………………………………………………………….xiii291H Publicationsand6H Awards…………………………………………………………….xvii29H Chapter7H 1. Introduction.............................................................................................1293H Chapter8H 2. Literature Review....................................................................................7294H 2.19H Toxicological Risk Assessment.................................................................................7295H 2.1.110H Hazard identification..........................................................................................10296H 2.1.21H Dose-response assessment .................................................................................11297H 2.1.2.112H Hormesis .....................................................................................................13298H 2.1.313H Exposure assessment..........................................................................................1529H 2.1.414H Risk characterisation ..........................................................................................1730H 2.215H Methods of Toxicity Testing...................................................................................18301H 2.2.116H Animal test methods...........................................................................................19302H 2.2.1.117H Criticism for the use of animal methods .....................................................2330H 2.2.218H Non-animal test methods ...................................................................................29304H 2.2.2.119H The Alternative movement..........................................................................29305H 2.2.2.220H In vitro toxicity endpoints ...........................................................................32306H 2.2.2.321H Mechanism of cellular toxicity....................................................................34307H 2.2.2.3.12H Basal cytotoxicity.......................................................................................................... 35308H 2.323H Validating In Vitro Tests and Regulatory Acceptance .......................................37309H 2.3.124H The MEIC/EDIT approach.................................................................................41310H 2.3.225H The ZEBET approach ........................................................................................43H 26H2.3.3 ICCVAM............................................................................................................44312H 2.3.427H ECVAM .............................................................................................................4431H 2.428H Using InVitro Cytotoxicity Tests in Risk Assessments.........................................45314H 2.529H Selected in vitro cytotoxicity assays .......................................................................49315H 2.5.130H The MTS cytotoxicity assay ..............................................................................49316H 2.5.231H The NRU cytotoxicity assay ..............................................................................51317H 2.5.332H The LDH cytotoxicity assay...............................................................................52318H v Table of Contents 2.5.43H The ATP cytotoxicity assay ...............................................................................53319H 2.634H Risk Assessment of Chemical Mixtures ................................................................54320H 2.6.135H Basic concepts of chemical mixture toxicology.................................................55321H 2.6.1.136H Simple similar action (Concentration Addition).........................................5532H 2.6.1.237H Simple dissimilar action (Response