Nanomaterial Hazard Identification

The Zebrafish Model for Rapid Material Testing

Research Project Proposal for Joe Fisher

Fall 2011












FDA hpf









APAC dpf





Animal Care and Use Protocol

Air Force Research Laboratory

Asia Pacific days post fertilization

Environmental Health and Safety

Engineered Nanomaterial(s)

Environmental Protection Agency

European Union

Food and Drug Administration hours post fertilization

Institutional Animal Care and Use Committee

Inductively Coupled Plasma Mass Spectroscopy

Instrumental Neutron Activation Analysis

National Institutes of Health

National Institute for Occupational Safety and Health

National Institute of Standards and Technology


National Science Foundation

Occupational Health and Safety

Oregon State University

Pacific Northwest National Laboratory

Research and Development

Registration, Evaluation, Authorization and Restriction of Chemicals

Sinnhuber Aquatic Research Laboratory

Toxic Substances Control Act

University of Oregon

United States


A. Introduction

The nanotechnology revolution has begun. The National Science Foundation predicts a shift from the first foundational phase (2001-2010) when the National Nanotechnology

Initiative (NNI) was launched, to the second foundational phase (2011-2020) that will include fundamentally new products based on nanomaterials (Roco et al., 2011). As these materials become ubiquitous in global commerce, very few of them have been tested for biocompatibility and safety. The issue with these new engineered materials is the uncertainty surrounding their risk to biological and environmental health.

In 1959 California Institute of Technology (Caltech) physicist Richard Feynman gave a talk titled “There's Plenty of Room at the Bottom” (Feynman, 1960) where he described the manipulation of elements at the atomic level. Thirty years later in 1989 scientists were able to specifically manipulate atoms when IBM arranged thirty-five atoms of xenon to form the letters “IBM”. Since that time global research and development (R&D) efforts led by the Americas; United States (US) and Canada, European Union (EU), and

Asia Pacific (APAC); Japan, Korea, Taiwan, China, have progressed the field of nanotechnology science. In year 2001, the US launched the NNI (Sargent, 2010) as a long term R&D program that coordinates nanotechnology related efforts among federal agencies. In 2004, the EU published its first strategy paper on nanotechnology (EESC,

2004). These programs and others propel the nanotechnology revolution forward.

Nanotechnology includes the manipulation of matter at the scale of individual atoms and molecules. One nanometer (1 x 10


meters) is about half the width of DNA and more than 1,000 times smaller than the width of a human red blood cell or 8,000 times smaller than the width of a human hair. The American Public Health Association issued policy stateme nt 20065 titled “Addressing Potential Environmental and Occupational

Health and Safety Risks of Nanotechnology” (APHA, 2006) that stated an urgent need to better understand and address health and safety issues surrounding nanotechnology.

Namomaterial regulation is progressing both in the European Union (EU) and United


States (US). Specific occupational and environmental health and safety regulations for nanomaterials will likely be seen in the near future. The movement to ensure safe use of nanomaterials will require testing and hazard identification models for this new type of material, as well as new methods to increase throughput in order to investigate the projected large number of new materials.

The purpose of this study is to investigate the zebrafish (

Danio rerio

) model as an organism for nanomaterial hazard identification. The zebrafish has become a popular model system to study vertebrate development and disease. It's current success can be traced back to the pioneering work of George Streisinger and his colleagues at the

University of Oregon (Walker et al., 1981; Stahl, 1995). The zebrafish is a small tropical freshwater fish. It is a vertebrate organism and therefore shares a high genomic, proteomic, and metabolomic homology with other vertebrates including a 80% homology with the human genome (Lieschke and Currie, 2007). The zebrafish is a whole organism that can provide more simultaneous system information than cell based studies. It is small, develops outside a maternal parent, and the embryo is optically transparent thus making it ideal for in-vivo studies in multiwell laboratory plates with light imaging and microscopic analysis. Compared to other laboratory animals, zebrafish fecundity is high and production and maintenance costs are relatively low. In response to the scientific community recommendations, the National Institutes of Health (NIH) has begun promoting zebrafish as a model organism for the study of vertebrate development and disease (Rasooly et al., 2003).

The study includes three aims. First, zebrafish as a model vertebrate organism is reviewed. Next, the zebrafish embryo heart rate is explored as a physiology end point.

Finally, nanomaterial biological adverse interactions with zebrafish embryos including morphology, physiology, behavior, and distribution are evaluated.


B. Study Purpose and Specific Aims

The purpose of this project is to evaluate the biocompatibility of a few nanomaterials.

New rapid material testing models are needed in order to evaluate the backlog of chemicals and nanomaterials entering the environment. The zebrafish model could potentially help fill a gap in throughput that currently exists in current chemical testing models and provide the needed platform for nanomaterial hazard identification. These new materials will also require the definition and use of additional parameters or ontologies to define the facts, dimensions, and relationships in the data. This project evaluates the relevancy and use of zebrafish as a model for nanomaterial testing. It fills a gap in the development of new data in agent testing and provides an addition to the science of health risk assessment.

Specific Aim 1: Review the literature and report on the zebrafish as a model vertebrate organism.

Research questions related to this aim are: Are zebrafish a toxicologically relevant platform for investigating nano-bio interactions? What endpoints

(morphology, physiology, behavior, biochemical, distribution) have been or could be utilized in analyzing zebrafish? How can zebrafish be used in high throughput screening systems to investigate the effects of exposure to potentially hazardous materials including nanomaterials?

Specific Aim 2: Review current zebrafish embryo heart rate assay methods and investigate the development of a novel high throughput method for conducting a zebrafish embryo heart rate assay.

Research questions related to this aim are: What current methods are available to conduct a zebrafish embryo heart rate assay? What methods are used on other vertebrate animals? Design and develop a prototype system for conducting a high throughput oriented heart rate assay on a zebrafish embryo.

Specific Aim 3: Test nano-bio interactions with zebrafish embryos to evaluate nanomaterial biocompatibility.

Research questions related to this aim are: What


phenotypes or endpoints for the developmental assays (morphology, physiology, behavior, and distribution) are observed that indicate an adverse biological effect? What biological fate can be detected or measured?

C. Background and Significance


1. There are currently a large number of untested chemicals (agents)

The vast number of untested chemicals in commerce is a significant public health concern. As such, the U.S. Environmental Protection Agency (EPA) asked the National

Research Council (NRC) to develop a long-range vision for toxicity testing in the 21st century (Tox-21c). The vision report by the National Academies of Science and National

Research Council, titled Toxicity Testing in the 21st Century: A Vision and a Strategy

(NRC, 2007) presents a new way of testing and risk assessment. The report and similar approaches by the EPA including ToxCast (Dix et al., 2007) provide vision and guidance on approaches for prioritizing the thousands of chemicals that need toxicity testing. Consisting of multi-federal agency collaboration including the EPA, FDA, and

NIH; Tox-21c plans to screen 10,000 chemicals by the end of 2011.

Much of the current US legislation on the safe use of chemicals is governed by the

Toxic Substance Control Act (TSCA) of 1976, the Federal Insecticide, Fungicide, and

Rodenticide Act (FIFRA) of 1972, the Food Quality Protection Act (FQPA) of 1996, and the Federal Food, Drug, and Cosmetics Act (FFDCA) of 1938. TSCA was considered to be the law that protected children, pregnant women, and the general population but has been ineffective since it requires the EPA to demonstrate the burden of proof

"unreasonable risk" of a chemical and not the producer of that chemical. In the thirty-five years since TSCA became law, less than 1% of the estimated 85,000 chemicals in commerce have been tested for safety. Recent calls for TSCA modernization from the

US Congress, industry, consumers, and others would require that chemical companies demonstrate the safety of their chemicals and allow EPA to evaluate that safety based


on the best available science. Legislative changes to TSCA could dramatically increase the amount of chemicals (agents) requiring testing including nanomaterials. Similar efforts in the European Union (EU) listed under the Registration, Evaluation,

Authorization and Restriction of Chemicals (REACH) legislation are already being implemented and will require a significant increase in testing during 2011-2017.

REACH, Tox-21c, and the new proposed TSCA reforms will significantly increase the requirement to test agents and investigate their potential impact on human and environmental health. Test requirements will further expand the need for alternative testing approaches including in vivo, in vitro, and in silico, in order to meet the increased need for agent testing.

2. Nanomaterials are new agents, soon ubiquitous, largely untested

Manufactured nanomaterials (NM), nanoparticles (NP), or ultrafine particles (UFP) particles are commonly defined as particles whose diameter is less than 0.1 um (< 100 nm). Manufactured NMs are new agents that will require toxicology testing and EPA will face challenges in regulating this risk (U.S. GAO, 2010). GAO estimates the world market for nanotechnology related products to total between $1 trillion and $2.6 trillion by 2015. The Project on Emerging Nanotechnologies currently estimates there are about 1,300 consumer nanomaterial containing products currently on the market (PEN,

2011). Products are categorized into 60% cosmetics and personal care products, 10% food and food packaging, and 30% all others. Nanomaterials present new challenges in testing because unlike most solid, liquid, or gas chemicals; nanomaterials present both physical and chemical properties (physiochemical) including size, shape, surface, and composition properties which often behave differently than bulk materials.

Concern for nanomaterial health and safety began to be published beginning with the report by the UK Royal Society and Royal Academy of Engineering (Dowling et al.,

2004). That same year a new subcategory of toxicology was proposed in a toxicology editorial titled Nanotoxicology (Donaldson et al., 2004). The following year an influential paper on Nanotoxicology was published (Obe rdörster et al,. 2005). Other important


papers continued to report on the research related to the health and safety concerns on nanomaterials. In the subsequent years two important journals 'Nature Nanotechnology'

(2006) and 'Nanotoxicology' (2007) began to publish important papers on nanomaterial health and safety.

3. Nanomaterials could be hazardous (they are different)

In 2009, the National Institute for Occupational Safety and Health published a report titled Approaches to Safe Nanotechnology (NIOSH, 2009) that listed guidelines for the safe use of nanomaterials in the workplace. The purpose of the report was to provide an overview about what is known about the hazards of nanomaterials and steps that can be taken to reduce workplace exposures. Potential health concerns cited in the report included; potential for nanomaterials to enter the body, enter the blood stream, translocate to organs, cause inflammation, and be toxic.

In theory nanomaterials can be produced from any element in the periodic table.

Elements of the periodic table are found in different phases or states defined by their properties; such as a solid, liquid, or gas. The majority of nanotechnology work for nanoparticles is focused on elements in the solid or phase at room temperature because they are easier to work with. Advances in the physical, chemical, and biological sciences as well as engineering and informatics have made nanotechnology a viable science.

4. In vivo testing is necessary in order to validate in vitro and in silico testing models

In vivo testing of nanomaterials is even more complex than traditional chemicals because of the added attributes of size, shape, and surface. In vivo nanomaterial testing needs to mature to the point where nanoparticles are tracked through portals of entry, then along pathways to systems or organs, and then as they are metabolized, stored, or excreted. This model of tracking nanomaterials is of course analogous to absorption, distribution, metabolism, and excretion (ADME) commonly used in pharmacokinetics and pharmacology. Clearly the best model to demonstrate nanomaterial biological


interactions in humans would be humans. The next best alternative would be any model that is homologous including animals with significant orthologs and paralogs to humans.

In vitro models have advantages and disadvantages when compared to in vivo models.

In vitro testing is important for mechanistic studies as it eliminates many of the confounding factors included with in vivo models. In vitro simplifies to a great extent the model being analyzed thus allowing a more focused analysis of mechanisms of action.

The disadvantage of in vitro studies is extrapolating the results back to in vivo models and humans.

In silico models are clearly the utopia of science. Something to be wished for, but never attained. Yet computational power and data storage continue to make significant advances. Processing capabilities continue to increase and costs continue to decrease.

Researchers continue to develop models of human and biological organism processes, organs, and cells. The development of structure activity relationship (SAR) models is one of the most popular techniques of in silico models.

In vivo, in vitro, and in silico models are the rule of systems biology. Blending the three elements together can ultimately increase the power of scientific studies and provide more weight of evidence for the effects of chemical, biological, and physical agents to biological and environmental health.


1. Zebrafish testing can be used to explore human health issues

The zebrafish is fast becoming a popular animal model for understanding disease, development, and toxicology in vertebrates. It along with other vertebrates including the human, mouse, and rat, can provide researchers with valuable information. The small size, fast development, optical clarity, and fecundity of the zebrafish make it easier and


more cost effective to work with than the human, mouse, and rat. Zebrafish are vertebrates with similar body plans and homology to humans. They are an obvious bridge between in vitro work and mammalian or human studies. The challenges of the immediate future will be to validate zebrafish models including assays, methods, and protocols.

The zebrafish genome assemblies are maintained by the Genome Reference

Consortium and the group is also the maintainer of the Human and Mouse genomes.

This bioinformatics data is used by a multitude of systems to compare and search the genomes. Work on zebrafish genome sequencing began in 2001 and the current zebrafish genome assemble, Zv9, was released in July 2010 (GRC, 2011). The next major assemble, Zv10, is expected in late 2012.

We can measure the morphological, developmental, biochemical, physiological, and behavior characteristics or anomalies of the zebrafish by utilizing a variety of phenotypes. Since the zebrafish develops ex-utero and fast, it's stages of development can easily be observed. The accessibility of the zebrafish embryo is one factor that makes it a powerful screening tool in the analysis of chemical, biological, or physical agents and their effect on the zebrafish organism.

2. Zebrafish testing could fill a huge gap in the backlog of agent testing

In 2004, the National Toxicology Program (NTP) published its vision and roadmap for the future. This vision and roadmap was later updated and published as the "Toxicity

Testing in the 21st Century: A Vision and a Strategy" (NAP, 2007). These landmark documents present the path forward for the U.S. toxicology program and all U.S. agencies concern with public health. The program now nicknamed as Tox21c proposes

"a shift from primarily in vivo animal studies to in vitro assays, in vivo assays with lower organisms, and computational modeling for toxicity assessments" (Collins et al., 2008).

From an in vivo perspective, zebrafish are considered a lower organism non mammalian model. This entire new direction in animal testing still revolves around the


3Rs guiding principals: Replacement, Reduction, and Refinement first described in 1959

(Russel et al., 1959). Many agencies are planning on funding research utilizing zebrafish to test chemical, biological, or physical agents. The EPA, FDA, and DoD are several examples. The Department of Defense (DoD) is currently planning to use

Zebrafish to examine the toxicology of nerve agents and the efficacy of associated therapeutic agents. Promising therapeutic agents would later be targeted for mammalian studies.

The zebrafish (Danio Rerio) fish was first reported scientifically by Francis Buchanan-

Hamilton (Hamilton, 1822) as a result of his survey work in and around the river Ganges on the Indian subcontinent. Since that time it has become a popular aquarium fish as well as a model vertebrate organism in scientific research. A literature review of 450 publications on the biology and use of zebrafish was conducted by Laale (1977).

Subsequent reviews or summaries have included husbandry (Lawrence, 2007), behavior and ecology (Spence et al, 2007), natural history (Engeszer et al, 2007), and toxicity (Embry et al, 2010). The zebrafish has been used to study chemical toxicity (Hill et al., 2005) and drug toxicity (Eimon and Rubinstein, 2009). It has also begun to be used in the study of nanomaterials (Isaacson et al., 2007) but further assay development and methods are needed. The utilization of the zebrafish organism could help reduce the existing backlog of untested chemicals and future test requirements as a result of REACH legislation (Hartung, 2010). Further testing is needed in order to establish zebrafish as a proven model for toxicity testing. In addition to the benefits of small size, the zebrafish transparency allows for improved in vivo optical imaging during agent testing. When combined with near infrared light (700-1000 nm), complete penetration and imaging of a zebrafish embryo and larvae are possible. The power of in vivo analysis in identifying hazardous materials is apparent. Dose response effects on living breathing organisms are more robust and generally more associative to human health effects than non complete organisms. The development of the zebrafish model for nanomaterial and other hazardous material testing can help bridge a gap between in vitro cell culture models and in vivo mammalian models (Fako and Furgeson, 2009).


3. Little current research demonstrates nanomaterial in vivo distribution and correlation to toxicity effect

A recent review on the biodistribution and toxicity of engineered gold nanoparticles

(GNP) determined that there are significant discrepancies in experimental conditions with in vitro and in vivo assays (Khlebtsov and Dykman, 2011). Khlebtsov and Dykman summarized the GNP in vivo biodistribution information published between 1995 and

2010 and found two swine, six rat, and twenty four mouse studies. The bulk of the research work was conducted between 2006 and 2010. Overall these studies represented a relatively small number of GNPs compared to the variety of GNPs being produced for biomedical applications. Most studies utilizing in vivo mammals are expensive and therefore sample sizes are small. Other reviewers cite that the development of nanoparticles for biomedical applications is currently expanding quickly yet very few have been tested for biocompatibility (Fadeel and Garcia-Bennett, 2010).


1. Develops new methods and/or assays for an in vivo model vertebrate organism

New assays, methods, and protocols continue to be developed for zebrafish. One of the first standard tests used to determine water quality or acute lethal toxicity (ISO 7346,

1996) was developed with zebrafish. OECD has published several tests utilizing fish including the recommended zebrafish for acute (OECD 203) and chronic (OECD 210,

212, 215) effects. OECD has published a draft guideline for the fish embryo toxicity test

(FET) with zebrafish being the recommended animal (Lammer et al., 2009). This draft

FET guideline is under further review and feedback is solicited. This work directly supports development of the FET and related zebrafish embryo assays.

2. Groundbreaking research on nanomaterial hazard identification and its correlation to toxicity effect in an in vivo model


As the global production of chemicals and nanomaterials continues to rise, new and innovative methods of testing these agents are needed. The research trend as recommended by Tox21c and REACH is to move from in vivo mammalian models to in vivo models of lower organisms, in vitro models, and in silico models. In vivo models utilizing the vertebrate zebrafish provide a platform that is capable of high throughput, statistical power, low material requirements, and direct interpretation to whole animal organisms. What the zebrafish model needs is more biocompatibility studies and further development of the assays, methods, and protocols used to conduct these studies. The testing of nanomaterials for biocompatibility is in its infancy stage, and much groundbreaking research is yet to be completed.

3. Provides a high throughput model for nanomaterial hazard identification

Many of the original ideas for nanomaterial high throughput screening originated from pharmaceutical in vitro high throughput screening systems. These systems utilize 96,

384, and 1536 well micro-titer plates. Cost savings and throughput considerations have resulted in the replacement of several human labor tasks for machine automation.

Robots are utilized for pick and place tasks. Liquid handlers automate sample delivery to micro-titer plates. Various other machines provide scientific analysis. This level of automation is common in large pharmaceutical companies, less common to smaller in vitro facilities, and very new to in vivo laboratories. Due to their small size and rapid development, zebrafish are one of the few in vivo animals compatible with these high throughput material handling methods including the use of 96 and 384 well micro-titer plates.

D. Materials and Methods

D. 1. Overview of Data Collection and Methods

This project will attempt to demonstrate the utility of the zebrafish model to investigate nano-bio interactions. Published literature, and the developing assays, methods, protocols, and databases will be investigated. Ontology and informatic parameters will


be researched from the developing standards. Zebrafish embryos will be obtained from the Oregon State University (OSU) Sinnhuber Aquatic Research Laboratory (SARL).

Nanomaterials with average diameters of less than 100 nm will be obtained from available resources. Aqueous exposure media will be produced and analyzed at available laboratories including OSU. Prototype assay methods will be explored at OSU.

Scientific equipment used to investigate nanomaterial biocompatiblity and fate will be utilized from OSU, the University of Oregon (UO), the Pacific Northwest National

Laboratory (PNNL).

D. 2. Description of Data Collection and Methods

Specific Aim 1: Review the literature and report on the zebrafish as a model vertebrate organism.

This aim will review the literature available on zebrafish as a model vertebrate organism. The search will include peer reviewed publications, government and university sources, conference materials, databases, and other non-peer reviewed but widely available literature. The specific aim is to provide evidence that zebrafish can be used for nanomaterial hazard identification and for the investigation of nano-bio interactions. This review will complement the rest of the project by laying a foundation for the analysis of a few nanomaterials.

Specific Aim 2: Review current zebrafish embryo heart rate assay methods and investigate the development of a novel high throughput method for conducting a zebrafish embryo heart rate assay.

Most vertebrate research animal testing includes physiology assays as a powerful indicator of agent effect. This aim will review the literature available on currently published heart rate assay methods. Then, the currently established video counting method will be reviewed and conducted utilizing SARL microscopic equipment. Next, prototype tools and equipment will be utilized to design and develop a novel high throughput compatible heart rate assay on a zebrafish embryo. This prototype system will utilize common off the shelf (COTS) semiconductor components and electronic


equipment. The aim is not to build a sophisticated system, but to build a prototype to demonstrate if future efforts would be a worthwhile investment.

Specific Aim 3: Test nano-bio interactions with zebrafish embryos to evaluate nanomaterial biocompatibility.

Animal testing conducted at the SARL is done in accordance with ACUP Number 3903 titled "Sinnhuber Aquatic Research Laboratory Stock Fish". These tests include several assays categorized as screening (morphological, physiological, and behavioral) and distribution. Due to insufficient microscope instrumentation at OSU, some imaging is conducted at the UO and PNNL. This off-site imaging is covered under the ACUP 3903

"12/30/2010" simple amendment which describes the transport and care of animals from SARL to UO or PNNL.

Experiments with zebrafish embryos are conducted in 96 well micro-titer plates using

100 ul of exposure media with one embryo per well. Developmental assays include 4 or

5 exposure levels and a minimum of one control. The experiments conducted will be the

SARL 5-day developmental embryo assay which includes morphological assessment at

1 and 5 dpf, behavior assessment at 1 dpf, and physiological assessment at 5 dpf.

Distribution assessments are conducted at 3 dpf to evaluate skin absorption or 5 dpf to evaluate gut absorption. Assessment includes up to; 20 attributes for morphology, 3 for behavior, and 2 for physiology. Distribution assessments are conducted via the gold standard INNA or ICP-MS methods, or via fluorescent labeling.

E. Timeline

Project Year 2011 - 2012

Specific Aim 1: Literature Review

Specific Aim 2: Heart Rate Assay

Specific Aim 3: Nano-bio Effect

Prepare Manuscript

Prepare Final Report

















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