Michelle R. Embry

The Fish Embryo Toxicity Test as an Animal Alternative Method in Hazard and Risk Assessment and Scientific Research

Animal alternatives research has historically focused on human safety assessments and has only recently been extended to environmental testing. This is particularly for those assays that involve the use of fish. A number of alternatives are being pursued by the scientific community including the fish embryo toxicity (FET) test, a proposed replacement alternative to the acute fish test. Discussion of the FET methodology and its application in environmental assessments on a global level was needed. With this emerging issue in mind, the ILSI Health and Environmental Sciences Institute (HESI) and the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) held an International Workshop on the Application of the Fish Embryo Test as an Animal Alternative Method in Hazard and Risk Assessment and Scientific Research in March, 2008. The workshop included approximately 40 scientists and regulators representing government, industry, academia, and non-governmental organizations from North America, Europe, and Asia. The goal was to review the state of the science regarding the investigation of fish embryonic tests, pain and distress in fish, emerging approaches utilizing fish embryos, and the use of fish embryo toxicity test data in various types of environmental assessments (e.g., hazard, risk, effluent, and classification and labeling of chemicals). Some specific key outcomes included agreement that risk assessors need fish data for decision-making, that extending the FET to include eluethereombryos was desirable, that relevant endpoints are being used, and that additional endpoints could facilitate additional uses beyond acute toxicity testing. The FET was, however, not yet considered validated sensu OECD. An important action step will be to provide guidance on how all fish tests can be used to assess chemical hazard and to harmonize the diverse terminology used in test guidelines adopted over the past decades. Use of the FET in context of effluent assessments was considered and it is not known if fish embryos are sufficiently sensitive for consideration as a surrogate to the sub-chronic 7-day larval fish growth and survival test used in the United States, for example. Addressing these needs by via workshops, research, and additional data reviews were identified for future action by scientists and regulators.

Critical analysis of literature on low-dose synergy for use in screening chemical mixtures for risk assessment

There is increasing interest in the use of tiered approaches in risk assessment of mixtures or co-exposures to chemicals for prioritization. One possible screening-level risk assessment approach is the threshold of toxicological concern (TTC). To date, default assumptions of dose or response additivity have been used to characterize the toxicity of chemical mixtures. Before a screening-level approach could be used, it is essential to know whether synergistic interactions can occur at low, environmentally relevant exposure levels. Studies demonstrating synergism in mammalian test systems were identified from the literature, with emphasis on studies performed at doses close to the points of departure (PODs) for individual chemicals. This search identified 90 studies on mixtures. Few included quantitative estimates of low-dose synergy; calculations of the magnitude of interaction were included in only 11 papers. Quantitative methodology varied across studies in terms of the null hypothesis, response measured, POD used to test for synergy, and consideration of the slope of the dose-response curve. It was concluded that consistent approaches should be applied for quantification of synergy, including that synergy be defined in terms of departure from dose additivity; uniform procedures be developed for assessing synergy at low exposures; and the method for determining the POD for calculating synergy be standardized. After evaluation of the six studies that provided useful quantitative estimates of synergy, the magnitude of synergy at low doses did not exceed the levels predicted by additive models by more than a factor of 4.

A European perspective on alternatives to animal testing for environmental hazard identification and risk assessment

Tests with vertebrates are an integral part of environmental hazard identification and risk assessment of chemicals, plant protection products, pharmaceuticals, biocides, feed additives and effluents. These tests raise ethical and economic concerns and are considered as inappropriate for assessing all of the substances and effluents that require regulatory testing. Hence, there is a strong demand for replacement, reduction and refinement strategies and methods. However, until now alternative approaches have only rarely been used in regulatory settings. This review provides an overview on current regulations of chemicals and the requirements for animal tests in environmental hazard and risk assessment. It aims to highlight the potential areas for alternative approaches in environmental hazard identification and risk assessment. Perspectives and limitations of alternative approaches to animal tests using vertebrates in environmental toxicology, i.e. mainly fish and amphibians, are discussed. Free access to existing (proprietary) animal test data, availability of validated alternative methods and a practical implementation of conceptual approaches such as the Adverse Outcome Pathways and Integrated Testing Strategies were identified as major requirements towards the successful development and implementation of alternative approaches. Although this article focusses on European regulations, its considerations and conclusions are of global relevance.

Creating context for the use of DNA adduct data in cancer risk assessment: I. Data organization.

The assessment of human cancer risk from chemical exposure requires the integration of diverse types of data. Such data involve effects at the cell and tissue levels. This report focuses on the specific utility of one type of data, namely DNA adducts. Emphasis is placed on the appreciation that such DNA adduct data cannot be used in isolation in the risk assessment process but must be used in an integrated fashion with other information. As emerging technologies provide even more sensitive quantitative measurements of DNA adducts, integration that establishes links between DNA adducts and accepted outcome measures becomes critical for risk assessment. The present report proposes an organizational approach for the assessment of DNA adduct data (e.g., type of adduct, frequency, persistence, type of repair process) in concert with other relevant data, such as dosimetry, toxicity, mutagenicity, genotoxicity, and tumor incidence, to inform characterization of the mode of action. DNA adducts are considered biomarkers of exposure, whereas gene mutations and chromosomal alterations are often biomarkers of early biological effects and also can be bioindicators of the carcinogenic process.

Adverse outcome pathways during early fish development: a conceptual framework for identification of chemical screening and prioritization strategies.

The fish early life-stage (FELS) test guideline (OECD 210 or OCSPP 850.1400) is the most frequently used bioassay for predicting chronic fish toxicity and supporting aquatic ecological risk assessments around the world. For each chemical, the FELS test requires a minimum of 360 fish and 1 to 3 months from test initiation to termination. Although valuable for predicting fish full life-cycle toxicity, FELS tests are labor and resource intensive and, due to an emphasis on apical endpoints, provide little to no information about chemical mode of action. Therefore, the development and implementation of alternative testing strategies for screening and prioritizing chemicals has the potential to reduce the cost and number of animals required for estimating FELS toxicity and, at the same time, provides insights into mechanisms of toxicity. Using three reference chemicals with well-established yet distinct adverse outcome pathways (AOPs) in early life stages of fish, we proposed FELS-specific AOPs as conceptual frameworks for identifying useful chemical screening and prioritization strategies. The reference chemicals selected as case studies were a cardiotoxic aryl hydrocarbon receptor agonist (2,3,7,8-tetrachlorodibenzo-p-dioxin), neurotoxic acetylcholinesterase inhibitor (chlorpyrifos), and narcotic surfactant (linear alkylbenzene sulfonate). Using qualitative descriptions for each chemical during early fish development, we developed generalized AOPs and, based on these examples, proposed a three-tiered testing strategy for screening and prioritizing chemicals for FELS testing. Linked with biologically based concentration-response models, a tiered testing strategy may help reduce the reliance on long-term and costly FELS tests required for assessing the hazard of thousands of chemicals currently in commerce.

Investigating Alternatives To The Fish Early-Life Stage Test: A Strategy For Discovering And Annotating Adverse Outcome Pathways For Early Fish Development

The fish early-life stage (FELS) test (Organisation for Economic Co-operation and Development [OECD] test guideline 210) is the primary test used internationally to estimate chronic fish toxicity in support of ecological risk assessments and chemical management programs. As part of an ongoing effort to develop efficient and cost-effective alternatives to the FELS test, there is a need to identify and describe potential adverse outcome pathways (AOPs) relevant to FELS toxicity. To support this endeavor, the authors outline and illustrate an overall strategy for the discovery and annotation of FELS AOPs. Key events represented by major developmental landmarks were organized into a preliminary conceptual model of fish development. Using swim bladder inflation as an example, a weight-of-evidence–based approach was used to support linkage of key molecular initiating events to adverse phenotypic outcomes and reduced young-of-year survival. Based on an iterative approach, the feasibility of using key events as the foundation for expanding a network of plausible linkages and AOP knowledge was explored and, in the process, important knowledge gaps were identified. Given the scope and scale of the task, prioritization of AOP development was recommended and key research objectives were defined relative to factors such as current animal-use restrictions in the European Union and increased demands for fish toxicity data in chemical management programs globally. The example and strategy described are intended to guide collective efforts to define FELS-related AOPs and develop resource-efficient predictive assays that address the toxicological domain of the OECD 210 test. Environ Toxicol Chem 2014;33:158–169.

A 21st century roadmap for human health risk assessment

Abstract The Health and Environmental Sciences Institute (HESI)-coordinated Risk Assessment in the 21st Century (RISK21) project was initiated to develop a scientific, transparent, and efficient approach to the evolving world of human health risk assessment, and involved over 120 participants from 12 countries, 15 government institutions, 20 universities, 2 non-governmental organizations, and 12 corporations. This paper provides a brief overview of the tiered RISK21 framework called the roadmap and risk visualization matrix, and articulates the core principles derived by RISK21 participants that guided its development. Subsequent papers describe the roadmap and matrix in greater detail. RISK21 principles include focusing on problem formulation, utilizing existing information, starting with exposure assessment (rather than toxicity), and using a tiered process for data development. Bringing estimates of exposure and toxicity together on a two-dimensional matrix provides a clear rendition of human safety and risk. The value of the roadmap is its capacity to chronicle the stepwise acquisition of scientific information and display it in a clear and concise fashion. Furthermore, the tiered approach and transparent display of information will contribute to greater efficiencies by calling for data only as needed (enough precision to make a decision), thus conserving animals and other resources.

Comparing laboratory and field measured bioaccumulation endpoints

An approach for comparing laboratory and field measures of bioaccumulation is presented to facilitate the interpretation of different sources of bioaccumulation data. Differences in numerical scales and units are eliminated by converting the data to dimensionless fugacity (or concentration-normalized) ratios. The approach expresses bioaccumulation metrics in terms of the equilibrium status of the chemical, with respect to a reference phase. When the fugacity ratios of the bioaccumulation metrics are plotted, the degree of variability within and across metrics is easily visualized for a given chemical because their numerical scales are the same for all endpoints. Fugacity ratios greater than 1 indicate an increase in chemical thermodynamic activity in organisms with respect to a reference phase (e.g., biomagnification). Fugacity ratios less than 1 indicate a decrease in chemical thermodynamic activity in organisms with respect to a reference phase (e.g., biodilution). This method provides a holistic, weight-of-evidence approach for assessing the biomagnification potential of individual chemicals because bioconcentration factors, bioaccumulation factors, biota-sediment accumulation factors, biomagnification factors, biota-suspended solids accumulation factors, and trophic magnification factors can be included in the evaluation. The approach is illustrated using a total 2393 measured data points from 171 reports, for 15 nonionic organic chemicals that were selected based on data availability, a range of physicochemical partitioning properties, and biotransformation rates. Laboratory and field fugacity ratios derived from the various bioaccumulation metrics were generally consistent in categorizing substances with respect to either an increased or decreased thermodynamic status in biota, i.e., biomagnification or biodilution, respectively. The proposed comparative bioaccumulation endpoint assessment method could therefore be considered for decision making in a chemicals management context.

Risk assessment in the 21st century: roadmap and matrix.

Abstract The RISK21 integrated evaluation strategy is a problem formulation-based exposure-driven risk assessment roadmap that takes advantage of existing information to graphically represent the intersection of exposure and toxicity data on a highly visual matrix. This paper describes in detail the process for using the roadmap and matrix. The purpose of this methodology is to optimize the use of prior information and testing resources (animals, time, facilities, and personnel) to efficiently and transparently reach a risk and/or safety determination. Based on the particular problem, exposure and toxicity data should have sufficient precision to make such a decision. Estimates of exposure and toxicity, bounded by variability and/or uncertainty, are plotted on the X- and Y-axes of the RISK21 matrix, respectively. The resulting intersection is a highly visual representation of estimated risk. Decisions can then be made to increase precision in the exposure or toxicity estimates or declare that the available information is sufficient. RISK21 represents a step forward in the goal to introduce new methodologies into 21st century risk assessment. Indeed, because of its transparent and visual process, RISK21 has the potential to widen the scope of risk communication beyond those with technical expertise.

Using mode of action information to improve regulatory decision-making: An ECETOC/ILSI RF/HESI workshop overview

The European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC), the International Life Sciences Institute (ILSI) Research Foundation (RF), and the ILSI Health and Environmental Sciences Institute (HESI) hosted a workshop in November 2009 to review current practice in the application of mode of action (MOA) considerations in chemical risk assessment. The aim was to provide a rationale for a more general, but flexible approach and to propose steps to facilitate broader uptake and use of the MOA concept. There was consensus amongst the workshop participants that it will require substantial effort and cooperation from the multiple disciplines involved to embrace a common, consistent, and transparent approach. Setting up a repository of accepted MOAs and associated guidance concerning appropriate data to support specific MOAs for critical effects would facilitate categorization of chemicals and allow predictions of toxicity outcomes by read-across. This should in future contribute to the reduction of toxicity testing in animals. The workshop participants also acknowledged the value and importance of human data and the importance of integrating information from biological pathway analyses into current MOA/human relevance frameworks.