Human and Ecological Risk Assessment, 13: 1164–1191, 2007 Copyright C Taylor & Francis Group, LLC ISSN: 1080-7039 print / 1549-7680 online DOI: 10.1080/10807030701655897
WORKSHOP REPORT Use of In Vitro Absorption, Distribution, Metabolism, and Excretion (ADME) Data in Bioaccumulation Assessments for Fish John Nichols,1 Susan Erhardt,2 Scott Dyer,3 Margaret James,4 Margo Moore,5 Kathleen Plotzke,6 Helmut Segner,7 Irvin Schultz,8 Karluss Thomas,9 Luba Vasiluk,10 and Annie Weisbrod3 1 National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Duluth, MN, USA; 2 Toxicology, Environmental Research and Consulting, Dow Chemical Company, Midland, MI, USA; 3 Central Product Safety, Procter and Gamble Company, Cincinnati, OH, USA; 4 Department of Medicinal Chemistry, University of Florida, Gainesville, FL, USA; 5 Department of Biological Sciences, Simon Fraser University, Burnaby, BC, Canada; 6 Health and Environmental Sciences, Dow Corning Corporation, Midland, MI, USA; 7 Centre for Fish and Wildlife Health, University of Bern, Bern, Switzerland; 8 Battelle, Pacific Northwest National Laboratory-Marine Research Operations, Sequim, WA, USA; 9 ILSI Health and Environmental Sciences Institute, Washington, DC, USA; 10 Department of Land Resource Science, University of Guelph, Guelph, ON, Canada ABSTRACT A scientific workshop was held in 2006 to discuss the use of in vitro Absorption, Distribution, Metabolism, and Excretion (ADME) data in chemical bioaccumulation assessments for fish. Computer-based (in silico) modeling tools are widely used to estimate chemical bioaccumulation. These in silico methods have inherent limitations that result in inaccurate estimates for many compounds. Based on a review of the science, workshop participants concluded that two factors, absorption and metabolism, represent the greatest sources of uncertainty in current bioaccumulation models. Both factors can be investigated experimentally using in vitro test systems. A variety Received 27 March 2007; revised manuscript accepted 1 April 2007. This article has been subjected to review by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. Address correspondence to John Nichols, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Duluth, MN 55804, USA. E-mail:
[email protected] 1164
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of abiotic and biotic systems have been used to predict chemical accumulation by invertebrates, and dietary absorption of drugs and xenobiotics by mammals. Research is needed to determine whether these or similar methods can be used to better predict chemical absorption across the gills and gut of fish. Scientists studying mammals have developed a stepwise approach to extrapolate in vitro hepatic metabolism data to the whole animal. A series of demonstration projects was proposed to investigate the utility of these in vitro–in vivo extrapolation procedures in bioaccumulation assessments for fish and delineate the applicability domain of different in vitro test systems. Anticipating research progress on these topics, participants developed a “decision tree” to show how in vitro information for individual compounds could be used in a tiered approach to improve bioaccumulation assessments for fish and inform the possible need for whole-animal testing. Key Words:
fish, bioaccumulation, bioconcentration, metabolism, biotransformation, absorption.
LIST OF ABBREVIATIONS ADME BAF BCF CEPA CDSL D7.4 GIT ILSI-HESI Kow OECD PAMPA PBiT POP QSAR REACH UNEP USEPA
Absorption, Distribution, Metabolism, Excretion Bioaccumulation Factor Bioconcentration Factor Canadian Environmental Protection Act Canadian Domestic Substances List n-octanol/phosphate buffer distribution coefficient at a pH of 7.4 Gastrointestinal Tract International Life Sciences Institute–Health and Environmental Sciences Institute n-octanol/water partition coefficient Organization for Economic Cooperation and Development Parallel Artificial Membrane Permeability Assay Persistent, Bioaccumulative, and inherently Toxic substance Persistent Organic Pollutant Quantitative Structure–Activity Relationship Registration, Evaluation, and Authorization of Chemicals Program (European Union) United Nations Environment Program U.S. Environmental Protection Agency
INTRODUCTION The accumulation of xenobiotics in fish and other aquatic biota is an issue of long-standing concern to industry, government regulators, the academic community, and the general public. Extensive research has been conducted to understand the chemical and biological processes that promote bioaccumulation, and detailed information is available for a small number of compounds, several of which are now banned from production and use. Increasingly, however, there is a need to Hum. Ecol. Risk Assess. Vol. 13, No. 6, 2007
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perform less intensive assessments for a much larger number of compounds. Regulatory programs in Europe and North America are being revised to support the 2004 enactment of the United Nations Stockholm Convention (also known as the Persistent Organic Pollutants (POPs) Protocol), which governs the assessment, use, trade, release, and replacement of all persistent (P), bioaccumulative (B), and inherently toxic substances (iT), or PBiTs (UNEP 2006). For example, the Canadian Environmental Protection Act (CEPA) required the Ministers of Environment and Health to categorize the hazard of approximately 23,000 chemicals on a Domestic Substances List (CDSL) and, as necessary, conduct screening level assessments to determine whether they are “. . . toxic or capable of becoming toxic to the environment or human health” (Government of Canada 1999; the word “toxic” is defined in Part 5, Section 64 of the Act). Legislation in Europe (Registration, Evaluation, and Authorization of Chemicals program; REACH) could result in similar reviews of tens of thousands of compounds (Rogers 2003). In most cases, these reviews are conducted in the absence of measured bioaccumulation data. Moreover, because of ethical concerns, many government agencies and animal welfare organizations are advocating large reductions in vertebrate testing, including testing with fish. These considerations suggest a need for alternative methods to assess the potential for chemicals to accumulate in fish. One method that is receiving considerable attention involves the use of in vitro test systems, alone or in combination with mathematical models. This report describes the results of a workshop held March 3–4, 2006, in San Diego, California, USA. Workshop participants were asked to review the state-of-thescience regarding the incorporation of in vitro Absorption, Distribution, Metabolism, and Excretion (ADME) information into bioaccumulation assessments for fish, and identify research needed to expand the utility and applications of this approach. An important outcome of this workshop was a proposal to conduct research on hepatic biotransformation in fish, with the goal of relating in vitro metabolic rate, in vivo metabolic rate, and measured levels of accumulation for a set of strategically selected compounds. Participants also discussed how in vitro data could be used in a tiered approach for bioaccumulation assessments. Based on this discussion, a “decision tree” was proposed to identify information required at each tier in the assessment process and provide guidance on the need for whole animal testing. The scope of the workshop was limited to consideration of in vitro methods that could be used to predict chemical accumulation in fish under standardized laboratory conditions. Participants recognized that a large number of factors may complicate efforts to predict accumulation in a natural setting, including the distribution of chemicals among environmental compartments, food web structure and function, and seasonal movements of animals. The extrapolation of bioaccumulation predictions from the lab to the field, and among different environmental settings, was identified as an important topic for future scientific workshops. Defining Bioconcentration and Bioaccumulation As applied to fish, the term bioconcentration refers to chemical accumulation that occurs in a waterborne exposure due to uptake across the gills and skin, whereas 1166
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the term bioaccumulation refers to chemical accumulation resulting from all possible routes of exposure, including dietary uptake. Bioconcentration is generally measured in controlled laboratory exposures, whereas bioaccumulation is typically characterized by measuring chemical concentrations in field-collected animals. The extent of bioconcentration may be expressed by calculating a bioconcentration factor (BCF; L/kg), which is the total chemical concentration in the animal (mg/kg) divided by that in water (mg/L). A bioaccumulation factor (BAF) with the same units can be developed to describe the extent of bioaccumulation. Alternatively, bioaccumulation may be referenced to the chemical concentration in sediment, resulting in a biota-sediment accumulation factor (BSAF). Unless otherwise indicated, BCFs, BAFs, and BSAFs represent the extent of accumulation that would be expected in a long-term exposure (i.e ., under steady-state conditions). These ratios are often normalized to the lipid content of fish and the freely dissolved chemical concentration in water (or, in the case of the BSAF, the lipid content of fish and the organic carbon content of sediment). The goal of this normalization is to account for differences in factors that control the uptake and accumulation of hydrophobic organic chemicals such as binding to organic material in water or sediment, and partitioning to tissue lipid. Bioaccumulation is the condition that results from a natural exposure, particularly for hydrophobic substances; however, BAFs and BSAFs are difficult to measure experimentally. As a result, regulators often use measured or modeled BCFs to estimate the potential for a compound to bioaccumulate, and legislated criteria for “bioaccumulation” are generally expressed as BCF values (Arnot and Gobas 2006). The major processes that determine the extent of chemical accumulation in fish are illustrated in Figure 1. Uptake processes include chemical absorption across the gills, skin, and gut. Loss processes include chemical efflux across the gills, skin, and gut; urinary and biliary elimination; and biotransformation. Growth affects the measured concentration of a chemical by increasing the tissue mass into which it is diluted. Additional processes are responsible for the internal distribution of a chemical. Among these are blood flow rates to individual tissues and tissue-specific differences in lipid content.
Current Methods to Estimate Bioaccumulation in Fish Three techniques are currently employed to assess the potential for a chemical to accumulate in fish: controlled exposures of fish to test chemicals in the water or diet, measurement of chemical residues in field-collected animals, and computational modeling. Measured in vivo accumulation data for fish are relatively scarce. For example, Arnot and Gobas (2006) reported that measured BCFs and BAFs are available for
