Advancing Actionable Alternatives to Vertebrate Animal Testing for Chemical Safety Assessment

Advancing Actionable Alternatives to Vertebrate Animal Testing for Chemical Safety Assessment is sponsored by U.S. Environmental Protection Agency (EPA). Supports research to develop alternatives to vertebrate animal testing for chemical safety assessments.

Advancing Actionable Alternatives to Vertebrate Animal Testing for Chemical Safety Assessment | US EPA Advancing Actionable Alternatives to Vertebrate Animal Testing for Chemical Safety Assessment U.S. Environmental Protection Agency National Center for Environmental Research Science to Achieve Results (STAR) Program CLOSED - FOR REFERENCES PURPOSES ONLY This is the initial announcement of this funding opportunity .

Funding Opportunity Number: EPA-G2018-STAR-C1 Catalog of Federal Domestic Assistance (CFDA) Number: 66. 509 Solicitation Opening Date: August 8, 2018 Solicitation Closing Date: September 25, 2018: 11:59:59 pm Eastern Time I. FUNDING OPPORTUNITY DESCRIPTION C.

Authority and Regulations D. Specific Areas of Interest/Expected Outputs and Outcomes III. ELIGIBILITY INFORMATION IV.

APPLICATION AND SUBMISSION INFORMATION A. Grants. gov Submittal Requirements and Limited Exception Procedures B.

Application Package Information C. Content and Form of Application Submission D. Submission Dates and Times F.

Submission Instructions and Other Submission Requirements V. APPLICATION REVIEW INFORMATION C. Past Performance History Review D.

Human Subjects Research Statement (HSRS) Review E. Additional Provisions for Applicants Incorporated into the Solicitation VI. AWARD ADMINISTRATION INFORMATION C.

Administrative and National Policy Requirements Access Standard STAR Forms ( How to Apply and Required Forms ) The U.S. Environmental Protection Agency (EPA), as part of its Science to Achieve Results (STAR) program, is seeking applications proposing research that will promote the development and use of alternative test methods and strategies that address the “3Rs” of toxicity testing: 1) reduce, 2) refine, and/or 3) replace vertebrate animal testing.

For the purposes of this RFA, alternative test methods refer to those that incorporate the “3Rs” principles. Pertinent research includes approaches such as analog/read-across techniques, mathematical models, and tiered testing approaches that integrate evidence from multiple sources to help accomplish these goals.

In addition to the development of new alternative test methods and strategies, translational science approaches that use available data to develop and/or advance actionable approaches for risk assessment of chemicals are also critical.

In this context, approaches that facilitate the use of existing animal data sources to reduce, refine, or replace the need for new vertebrate animal tests are as welcome as those that provide new data streams.

The research activities to be funded under this announcement are intended to advance the science underpinning the use of non-vertebrate test methods, and to develop actionable alternative approaches to: 1) developmental toxicity tests in humans; 2) reproductive toxicity tests in humans; and/or 3) ecotoxicity tests.

This solicitation provides the opportunity for the submission of applications for projects that may involve human subjects research. Human subjects research supported by the EPA is governed by EPA Regulation 40 CFR Part 26 (Protection of Human Subjects). This includes the Common Rule at subpart A and prohibitions and additional protections for pregnant women and fetuses, nursing women, and children at subparts B, C, and D.

Research meeting the regulatory definition of intentional exposure research found in subpart B is prohibited by that subpart in pregnant women, nursing women, and children. Research meeting the regulatory definition of observational research found in subparts C and D is subject to the additional protections found in those subparts for pregnant women and fetuses (subpart C) and children (subpart D).

All applications must include a Human Subjects Research Statement (HSRS, as described in Section IV. C. 5.

c of this solicitation), and if the project involves human subjects research, it will be subject to an additional level of review prior to funding decisions being made as described in Sections V. D and V. E of this solicitation.

Guidance and training for investigators conducting EPA-funded research involving human subjects may be obtained here: Basic Information about Human Subjects Research Basic EPA Policy for Protection of Subjects in Human Research Conducted or Supported by EPA The STAR Program’s goal is to stimulate and support scientific and engineering research that advances EPA’s mission to protect human health and the environment.

It is a competitive, peer-reviewed, extramural research program that provides access to the nation’s best scientists and engineers in academic and other nonprofit research institutions. STAR funds research on the environmental and public health effects of air quality, environmental changes, water quality and quantity, hazardous waste, toxic substances, and pesticides.

Anticipated Type of Award: Grant or Cooperative Agreement Estimated Number of Awards: Approximately five awards Anticipated Funding Amount: Approximately $4,250,000 total for all awards Potential Funding per Award: Up to a total of $850,000, including direct and indirect costs, with a maximum duration of 3 years. Cost-sharing is not required. Proposals with budgets exceeding the total award limits will not be considered.

Public and private nonprofit institutions/organizations, public and private institutions of higher education, and hospitals located in the U.S., state and local governments, Federally Recognized Indian Tribal Governments, and U.S. territories or possessions are eligible to apply. See full announcement for more details. To apply under this solicitation, use the application package available at Grants.

gov (for further submission information see Section IV. F. “Submission Instructions and other Submission Requirements”).

Note: With the exception of the current and pending support form, all necessary forms are included in the electronic application package ( How to Apply and Required Forms ). Make sure to include the current and pending support form in your Grants. gov submission.

If your organization is not currently registered with Grants. gov, you need to allow approximately one month to complete the registration process. Please note that the registration process also requires that your organization have a unique entity identifier (e.g., ‘DUNS number’) and a current registration with the System for Award Management (SAM) and the process of obtaining both could take a month or more.

Applicants must ensure that all registration requirements are met in order to apply for this opportunity through Grants. gov and should ensure that all such requirements have been met well in advance of the submission deadline. This registration, and electronic submission of your application, must be performed by an authorized representative of your organization.

If you do not have the technical capability to utilize the Grants. gov application submission process for this solicitation, see Section IV. A below for additional guidance and instructions.

Technical Contact: Mitch Lasat (lasat. mitch@epa. gov); phone: 202-564-7702; Dan Villeneuve ( villeneuve.

dan@epa. gov) ; phone: 218-529-5217 Eligibility Contact: Ron Josephson (josephson. ron@epa.

gov); phone: 202-564-7823 Electronic Submissions: Debra M. Jones (jones. debram@epa.

gov); phone: 202-564-7839 I. FUNDING OPPORTUNITY DESCRIPTION A more efficient, predictive, and economical system for assessing adverse health effects of chemical substances was envisioned in the seminal National Research Council (NRC) report, Toxicity Testing in the 21st Century: A Vision and a Strategy (NRC 2007).

Part of the motivation for this strategy involved Reduction in the total amount of vertebrate animal testing, Refinement of animal testing methods to lessen pain and distress, and ultimately Replacement of vertebrate animal testing with scientifically-sound alternatives wherever possible.

These have been termed the “3Rs” of toxicity testing and have been hallmark principles in defining alternative test methods for over 50 years (Russell and Burch, 1959).

While the NRC report (NRC 2007) was focused on human health, it has been recognized that alternative testing strategies that incorporate the “3Rs” principles can support more efficient, predictive, and economical approaches to assessing human health and ecological risk alike.

Consequently, many stakeholders within the regulatory community have been working in coordination with the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), the National Toxicology Program’s Center for the Evaluation of Alternative Toxicological Methods (NICEATM), the Organization for Economic Cooperation and Development (OECD), the European Chemicals Agency (ECHA), Canadian environmental authorities, and a range of other agencies, institutes, and programs to develop, validate, and implement alternative approaches for chemical safety assessment.

Terminology - for the purposes of this RFA: In vitro – refers to assays conducted outside of an intact animal, for example in a tube or multi-well plate. Examples would include cell-based assays, cell-free biochemical assays, or those employing organotypic cells, spheroids, organoids, etc. In silico – refers to approaches employing computer simulation and computation rather than biological systems.

Examples include structure-activity relationships, physiologically-based models, agent-based tissue development and differentiation models, etc. A lternative test methods - refer to those that incorporate the “3Rs” principles (Russell and Burch, 1959).

Strategies include approaches such as analog/read-across techniques, mathematical models, and tiered testing approaches that integrate evidence from multiple sources to help accomplish those same goals. Translational research - refers to moving from research on development of a new assay, model, or strategy toward practical implementation and use for chemical safety evaluations and associated risk-based decision-making.

Regulatory -refers mainly to activities beneficial to states and industry. The proposed research is not intended to be for the direct benefit or use of federal regulatory entities.

This effort draws upon decades of experience within the toxicological research community developing in vitro assays as well as building and testing computational models ( in silico approaches) that can evaluate the ability of a chemical, or more complex mixtures (e.g., formulated products, environmental samples) to act on specific biological pathways or elicit non-specific stress responses (e.g., cytotoxicity).

Many such assays and models can be implemented in a high throughput format as demonstrated through efforts like ToxCast (Kavlock et al. 2012) and Tox21 (Tice et al. 2013).

However, in many cases the use of data from alternative tests in risk assessment has been limited by the lack of translational science that clearly defines what the assay or computational model is measuring or predicting and, even more importantly, what the data mean to potential human health or ecological effects.

Acceptance of alternative methods that support the “3Rs” principles often requires that the alternative methods can be shown to be “scientifically reliable, relevant, and capable of providing information of equivalent or better scientific reliability and quality to that which would be obtained from vertebrate animal testing” (TSCA 2016, Section 4(h)(2)(A)).

In this context, reliability refers to the extent of reproducibility of results from tests performed within and among laboratories over time. It is largely determined by establishing and evaluating assay performance criteria. Relevance, on the other hand, concerns the extent to which the test, and resulting data, are useful for addressing regulatory needs.

The adverse outcome pathway (AOP) framework (Ankley et al. 2010; Villeneuve et al. 2014; OECD AOP Programme) has been developed as a means to organize and evaluate scientific evidence supporting the relevance and use of alternative methods for predicting or inferring outcomes of regulatory significance.

Information captured in AOP descriptions can be further complemented by empirical assay or computational model descriptions that clearly define the scope, limitations, and domain(s) of applicability in which the results can be interpreted and/or extrapolated (i.e., providing transparency with regard to the methodology and the associated assumptions and limitations).

A major goal of this RFA is to advance development and use of alternative test methods and strategies to reduce, refine, and/or replace vertebrate animal testing. While the present solicitation invites development of new alternative test methods and strategies, translational science that makes data from those approaches actionable in a risk assessment context is critical.

In that context, projects that establish the relevance, reliability, and scientific quality of existing alternatives, relative to current vertebrate testing approaches, in a way that can facilitate the acceptance and uptake in risk assessment and decision-making, will be regarded just as favorably as those that involve the de novo development of new in vitro assays or in silico approaches.

To the extent possible, proposals involving the development of novel alternative test methods or strategies should also emphasize the translational science needed to facilitate its use in risk-based decision-making. EPA recognizes that it is important to engage all available minds to address the environmental challenges the nation faces.

At the same time, EPA seeks to expand the environmental conversation by including members of communities which may have not previously participated in such dialogues to participate in EPA programs. For this reason, EPA strongly encourages all eligible applicants identified in Section III, including minority serving institutions (MSIs), to apply under this opportunity.

For purposes of this solicitation, the following are considered MSIs: Historically Black Colleges and Universities, as defined by the Higher Education Act (20 U.S.C. § 1061). A list of these schools can be found at White House Initiative on Historically Black Colleges and Universities ; Tribal Colleges and Universities, as defined by the Higher Education Act (20 U.S.C.

§ 1059(c)). A list of these schools can be found at American Indian Tribally Controlled Colleges and Universities; Hispanic-Serving Institutions (HSIs), as defined by the Higher Education Act (20 U.S.C. § 1101a(a)(5)).

There is no list of HSIs.

HSIs are institutions of higher education that, at the time of application submittal, have an enrollment of undergraduate full-time equivalent students that is at least 25% Hispanic students at the end of the award year immediately preceding the date of application for this grant; and Asian American and Native American Pacific Islander-Serving Institutions; (AANAPISIs), as defined by the Higher Education Act (20 U.S.C. § 1059g(a)(2)).

There is no list of AANAPISIs. AANAPISIs are institutions of higher education that, at the time of application submittal, have an enrollment of undergraduate students that is not less than 10 % students who are Asian American or Native American Pacific Islander. Alternatives to vertebrate animal testing have been routinely used in chemical safety-related decision-making for many years.

For example, baseline aquatic toxicity is routinely predicted from physicochemical properties using ECOSAR (ECOSAR 2018). OncoLogic TM (Oncologic, 2018) is an in silico tool used to predict potential to cause cancer in humans.

More recently, alternative testing methods suitable for evaluating skin sensitization, eye and skin irritation, and mutagenic potential are proceeding through an ICCVAM and/or OECD validation process to provide the basis for acceptance as reliable and relevant alternatives to vertebrate animal sensitization and irritation tests (Strickland et al. 2016; Ezendam et al. 2016).

Consequently, precedence for successful development and implementation of alternatives to vertebrate testing and their use in chemical safety assessment exists. Nonetheless, there remain many toxicological outcomes, and associated test guidelines, pertinent to chemical safety assessment for which alternatives to vertebrate testing have not been sufficiently developed to facilitate use in risk-based decision-making.

For example, repeat oral dosing studies with rodents (e.g., OECD test guideline [TG] 407), which involve conducting histopathology on over 40 different tissues, represent a daunting challenge for the development of alternatives. Thus, efforts are being undertaken to identify the effects most frequently observed in those tests as a basis to prioritize development of viable alternatives.

In the nearer term, perhaps more tractable, yet critical, opportunities for the development of alternatives to vertebrate animal tests are in the area of assessing developmental and reproductive toxicity (DART; Sciali et al. 2018). Developmental and reproductive toxicity tests are both costly and time-consuming, and employed in a wide range of risk assessments.

Consequently, development of viable non-vertebrate alternatives to the prenatal developmental toxicity test (e.g., OECD test guideline [TG] 414), reproductive and developmental toxicity test (e.g., OECD TG 421), and the one and two generation reproductive toxicity tests (e.g., OECD TG 415 and 416) and related vertebrate DART methods would be broadly useful.

Ecological risk assessments also employ vertebrate tests for which alternative test methods and strategies are desired (e.g., Volz et al. 2011). As noted earlier, structure-activity relationship-based approaches have long been utilized for predicting so called “baseline” acute and chronic toxicity to aquatic organisms.

However, for pathways of toxicity other than narcosis, ecological hazard assessment typically still relies on vertebrate ecotoxicity tests to evaluate chemicals for specific chemical modes of action (e.g., neurotoxicity, immune modulations, teratogenesis, endocrine disruption). Stinckens et al.

(2018) recently showed the utility of cell-free biochemical assays, anchored to an AOP, for predicting adverse effects on swim bladder development in fish. However, this is just one example of an alternative test method that might be integrated into an overall strategy for refining and reducing the need for vertebrate ecotoxicity tests.

Examples of tests for which alternatives are lacking include; fish early life stage test (OECD TG 210), fish juvenile growth test (OECD TG 215), 21-d fish short term reproduction assay (OECD TG 229), fish sexual development test (OECD TG 234), avian reproduction test (OECD TG 206), and avian dietary test (OECD 205).

Consequently, within both the human health and ecological toxicity arenas, there are opportunities for development of alternative test methods and strategies that can help implement 3Rs principles into risk-based decision making.

Developing translational science that demonstrates the reliability and relevance of these approaches and shows how they can be practically employed to provide chemical safety assessments that are of equivalent or better scientific quality relative to current vertebrate tests can help to facilitate implementation of the vision for the use of 21 st century science in risk-based evaluation of chemicals (NAS 2017). C.

Authority and Regulations The authority for this RFA and resulting awards is contained in the Toxic Substances Control Act, 15 U.S.C. 2609, Section 10 and Federal Insecticide, Fungicide, and Rodenticide Act, 7 U.S.C. 136r, Section 20.

For research with an international aspect, the above statutes are supplemented, as appropriate, by the National Environmental Policy Act, Section 102(2)(F). Note that a project’s focus is to consist of activities within the statutory terms of EPA’s financial assistance authorities; specifically, the statute(s) listed above.

Generally, a project must address the causes, effects, extent, prevention, reduction, and elimination of air pollution, water pollution, solid/hazardous waste pollution, toxic substances control, or pesticide control depending on which statute(s) is listed above. Further note applications dealing with any aspect of or related to hydraulic fracking will not be funded by EPA through this program.

Additional applicable regulations include: 2 CFR Part 200, 2 CFR Part 1500, and 40 CFR Part 40 (Research and Demonstration Grants). D. Specific Areas of Interest/Expected Outputs and Outcomes Note to applicant: The term “output” means an environmental activity, effort, and/or associated work products related to an environmental goal or objective, that will be produced or provided over a period of time or by a specified date.

The term “outcome” means the result, effect or consequence that will occur from carrying out an environmental program or activity that is related to an environmental or programmatic goal or objective. EPA requires that grant applicants adequately describe environmental outputs and outcomes to be achieved under assistance agreements (see EPA Order 5700. 7A1, Environmental Results under Assistance Agreements ).

Applicants must include specific statements describing the environmental results of the proposed project in terms of well-defined outputs and, to the maximum extent practicable, well-defined outcomes that will demonstrate how the project will contribute to the priorities described above.

The research activities to be funded under this announcement are intended to advance the science underpinning the use of non-vertebrate alternative test methods, and/or strategies based on such methods, in chemical hazard assessment.

The emphasis is on development of the translational science needed to interpret and utilize in vitro , in silico , or non-vertebrate animal test results to inform hazard assessments that have traditionally relied on vertebrate animal testing. The desired outcome of this solicitation is the reduction of vertebrate animal testing and its eventual replacement by efficient and cost effective alternative methodologies.

To that end, applicants are encouraged to consider the 3Rs in the development and implementation of the proposed research. Targeted use of legacy in vivo data or new approaches to obtain mechanistic data from archived tissue samples (e.g., Wehmans et al. 2018) for verification is encouraged.

While some studies with vertebrates may be needed to validate and establish the relevance, predictive utility, and/or scientific quality of the alternative approach or strategy proposed, use of vertebrate animals in the research should be minimized and existing vertebrate test data should be leveraged to achieve the research objectives wherever scientifically feasible.

In developing the proposals, applicants should address at least one of the three research areas described below. Proposals may respond to one research area in detail or may integrate their work across multiple research areas.

For example, to the extent that vertebrate biology is conserved, certain alternative tests or strategies, and the associated science-based interpretive tools (e.g., assays aligned with key events in AOPs, evidence and relationships that support extrapolation from in vitro to in vivo, across levels of biological organization, or across species, computational models, etc.) may lend themselves to a “One Health” (Buttke, 2011) approach in which the same alternative tests or strategies may be viable for reducing, refining, or replacing both health and ecology focused tests.

Proposals should clearly indicate which research area(s) is being addressed. Proposals that address more than one research area will not necessarily be rated more highly than those that address just one area. Specific Research Areas : Non-vertebrate alternatives to human health-related developmental toxicity tests.

Research in this area should be aimed at establishing the relevance and reliability of one or more approaches or strategies that employ in vitro, in silico, and/or non-vertebrate animal test methods to provide information scientifically equivalent to that which can be obtained from a prenatal developmental toxicity test (e.g., OECD TG 414), or related/similar tests focused on detecting potential hazards to human development.

Non-vertebrate alternatives to human health-related reproductive toxicity tests.

Research in this area should be aimed at establishing the relevance and reliability of one or more approaches or strategies that employ in vitro, in silico, and/or non-vertebrate animal test methods to provide information scientifically equivalent to that which can be obtained from a reproductive and developmental toxicity test (e.g., OECD TG 421), or one and two generation reproductive toxicity tests (e.g., OECD TG 415 and 416), or related/similar tests employing vertebrate animals to detect potential hazards to human reproduction.

Non-vertebrate alternatives to vertebrate ecotoxicity tests.

Research in this area should be aimed at establishing the relevance and reliability of one or more novel approaches or strategies that employ in vitro, in silico, and/or non-vertebrate methods to provide information scientifically equivalent to that which can obtained from ecotoxicity testing guidelines employing vertebrate animal models such as the fish early life stage test (e.g., OECD TG 210), fish juvenile growth test (e.g., OECD TG 215), 21-d fish short term reproduction assay (e.g., OECD TG 229), fish sexual development test (e.g., OECD TG 234), the avian reproduction test (e.g., OECD TG 206), or the avian dietary test (e.g., OECD 205), or related/similar tests.

Research in this area should focus on ecotoxicology outcomes other than baseline narcosis toxicity which is routinely predicted from chemical structure using ECOSAR (ECOSAR 2018).

Note on non-vertebrate animal tests: Tests conducted with intact, non-vertebrate, organisms (e.g., planaria, hydra, drosophila, crustaceans, etc.), particularly those amenable to high throughput, are considered as alternative test methods for the purpose of this RFA.

Tests conducted with intact vertebrate organisms, even at early life stages and/or performed in high throughput (e.g., fish embryo tests) are NOT considered alternatives for the purposes of this RFA.

For the purposes of this RFA, EPA is not interested in supporting research on development of alternatives to vertebrate tests employed in endocrine disruptor screening (EDSP, 2018), i.e., focused on detecting agonism or antagonism of estrogen receptors, androgen receptors, inhibition of steroidogenesis, or disruption of the thyroid axis.

Applicants proposing research on the development of alternatives to vertebrate tests employed in endocrine disruptor screening would not be considered responsive to the research needs of this RFA (see peer review criterion #2 below). Regardless of the specific research area(s) selected, applicants should address a similar set of scientific objectives.

To the extent possible, the proposed research should address all four objectives outlined below: Objective A. Identification of one or more non-vertebrate animal methods that can be used to reduce, refine, or replace the need to conduct one or more vertebrate toxicity tests as identified in the specific research areas above.

This can include development of new alternative test methods or strategies or refinements/improvements of existing alternatives and strategies.

Refinements/improvements may include incorporation of alternatives into an integrated testing and assessment approach (IATA; OECD 2017) or similar strategy that allows for a suite of assays and models to be used as a scientifically reliable and relevant alternative to a specific in vivo vertebrate test.

For example, complex in vitro (i.e., biomimetic microsystems) and in silico (i.e., computational systems models) methods that recapitulate organotypic structure and function could be used for assessing relevant key events in AOP-based IATAs. Applicants should clearly anchor their alternative method(s) and/or strategy to one or more apical endpoints of accepted relevance to risk-based decision-making.

This is best achieved through alignment with established endpoints in OECD approved animal toxicity test guidelines (examples provided above). Applicants are also encouraged to consult the OECD Guidance Document on Validation and International Acceptance of New or Updated Test Methods for Hazard Assessment (OECD 2005).

While formal OECD or ICCVAM validation of the alternative test method(s) or strategy are beyond the scope of this solicitation, incorporation of research elements that would help facilitate and potentially accelerate formal validation is encouraged.

To the extent possible, new alternative methods developed as part of the proposed research should be documented in a manner consistent with OECD recommendations for describing non-guideline in vitro tests (OECD 2014) or similar guidance for in silico methods. If the alternative approach or strategy employs existing data sources (e.g., ToxCast), similar assay descriptions should be obtained or developed as part of the research effort.

Objective B. Development of interpretive context that facilitates application of the alternative approach or strategy in risk-based decision-making. Scientific evidence supporting the relevance of the alternatives for predicting apical in vivo outcomes should be organized and documented using the AOP framework.

To the extent possible, the information should be deposited in the AOP knowledgebase (aopwiki. org) as part of the project (OECD 2016; Horvat et al. 2017; Song et al.

2017; Fay et al. 2017). The interpretive context should also include relevant mathematical or computational prediction models, that can be used to quantitatively or semi-quantitatively infer the probability or severity of apical, adverse, outcomes from the data provided via the alternative approach(es) or strategy.

This could include definition of computational approaches for in vitro to in vivo extrapolation (IVIVE) under acute, episodic, or chronic exposure scenarios.

It may also entail measurement or estimation of assay-specific parameters (e.g., in the case of an in vitro bioassay – volume of solution per well, protein and lipid content, assay plate composition, etc.) needed to estimate free versus bound chemical fractions in the test system (as appropriate) to facilitate extrapolation of effect concentrations measured in the alternative assay to equivalent in vivo doses (e.g., concentrations or plasma or relevant tissue compartments in which key events in an AOP are measured) or associated external exposure (e.g., food, water, air) concentrations (e.g., Fischer et al.

2017). Interpretive context could also include approaches for cross-species interpretations and extrapolations of the results and estimating potential differences in species sensitivities. Objective C.

Conducting one or more case studies that evaluate the potential of alternative strategies and/or methods to replace vertebrate animal toxicity tests.

Case studies should include development of (a) a generalizable application workflow, (b) performance-based quality metrics and associated data evaluation and acceptance criteria, (c) identification of suitable positive and negative reference chemicals (where appropriate), and (d) associated presentations and tutorials adequate to assure that interested stakeholders can be properly trained to understand and use the alternative method(s) and/or strategy.

Development of a generalized application workflow may take the form of an IATA (OECD 2017) in which expert judgement is employed to integrate the different types of evidence generated via the alternative or strategy. Alternatively, more structured rule-based approaches for fixed interpretation of results from the information sources (termed a defined approach) may also be developed.

Whatever the form, a systematic and transparent approach for interpreting and using the results in a decision-making context should be demonstrated.

In cases where the application workflow is facilitated via development of novel IT implementation, the workflow should be explicit regarding source of foundational data, notably what data were used and how, providing meta-data that includes specification of reporting standards, data formats, terminology, curation and repurposing of data; and adhere to FAIR (Findable, Accessible, Interoperable, and Reusable) Principles ( The road to FAIR: FAIR principles for the Social Sciences and Humanities.

. Objective D. Documented analysis of the value of information obtained from the alternative method(s) or strategy for reducing, refining, or replacing the need to conduct one or more vertebrate toxicity tests as appropriate to the specific research area(s).

To the extent feasible, this should provide critical evaluation of whether the alternative method(s) or strategy provides information of equivalent or better quality relative to the associated guideline vertebrate test(s) and potential limitations of the information.

Additionally, throughput and cost of data collection and analysis should be estimated based on the generalized workflows developed through case studies, along with reasonable and documented assumptions concerning scale up of the alternative method(s) or strategy from research and development to anticipated routine use.

Expected Outputs and Outcomes: Outputs expected from this solicitation may include publications of research results in peer-reviewed journals, guidance documents, decision support tools, models, software applications that implement a workflow, demonstration and case studies, reports, AOP descriptions submitted to the AOP-Wiki (aopwiki. org) and presentations.

Scientific objectives under the specific research areas defined above may be addressed as independent outputs or integrated into one or more overarching outputs. Completion of the outputs defined should lead to outcomes such as improving the ability to rapidly identify, establish confidence in, and implement additional alternative test methods and strategies into chemical risk assessment practices.

The proposed research should serve the long-term outcome of reducing and eventually replacing the need for selected vertebrate animal tests currently employed routinely in chemical hazard and/or risk assessment. Ankley GT, Bennett RS, Erickson RJ, Hoff DJ, Hornung MW, Johnson RD, Mount DR, Nichols JW, Russom CL, Schmieder PK, Serrrano JA, Tietge JE and Villeneuve DL.

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