Approaches and considerations for the assessment of immunotoxicity for environmental chemicals: a workshop summary.

As experience is gained with toxicology testing and as new assays and technologies are developed, it is critical for stakeholders to discuss opportunities to advance our overall testing strategies. To facilitate these discussions, a workshop on practices for assessing immunotoxicity for environmental chemicals was held with the goal of sharing perspectives on immunotoxicity testing strategies and experiences, developmental immunotoxicity (DIT), and integrated and alternative approaches to immunotoxicity testing. Experiences across the chemical and pharmaceutical industries suggested that standard toxicity studies, combined with triggered-based testing approaches, represent an effective and efficient approach to evaluate immunotoxic potential. Additionally, discussions on study design, critical windows, and new guideline approaches and experiences identified important factors to consider before initiating DIT evaluations including assay choice and timing and the impact of existing adult data. Participants agreed that integrating endpoints into standard repeat-dose studies should be considered for fulfilling any immunotoxicity testing requirements, while also maximizing information and reducing animal use. Participants also acknowledged that in vitro evaluation of immunosuppression is complex and may require the use of multiple assays that are still being developed. These workshop discussions should contribute to developing an effective but more resource and animal efficient approach for evaluating chemical immunotoxicity.

Juvenile immunotoxicology.

Developmental immunotoxicity (DIT) testing is centered around the concern that exposure to immunotoxicants early in development may result in enhanced susceptibility of, or unique or more persistent effects on, the immune system, in comparison to adult exposure. Developmental immunotoxicity has been the focus of numerous workshops and reviews for at least fifteen years. Most of these earlier activities have focused on both environmental chemicals and pharmaceuticals and have concluded that the best approach to DIT is to address the possible impacts of exposure during all of the critical windows of development. This article will emphasize the critical role played by exposure during the juvenile stage of development. This article will also highlight several key issues that distinguish DIT testing of pharmaceuticals. Representatives from the pharmaceutical, biotechnology, academic, and regulatory sectors (both FDA and EMA) were brought together during a two-day workshop in May 2010 to consider the current state of the science of DIT as it pertains to the testing of pharmaceuticals. It is important to emphasize at the onset that there are currently no regulatory guidelines for either drugs or nondrug chemicals specifically focused on assessment of DIT, although some general guidelines are included in both developmental and reproductive toxicity and general immunotoxicology guidance documents.

Human and environmental health challenges for the next decade (2010–2020).

The public health and environmental communities will face many challenges during the next decade. To identify significant issues that might be addressed as part of the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) scientific portfolio, an expert group of key government, academic, and industry scientists from around the world were assembled in 2009 to map the current and future landscape of scientific and regulatory challenges. The value of the scientific mapping exercise was the development of a tool which HESI, individual companies, research institutions, government agencies, and regulatory authorities can use to anticipate key challenges, place them into context, and thus strategically refine and expand scientific project portfolios into the future.

Predicting future human and environmental health challenges: the Health and Environmental Sciences Institute's scientific mapping exercise.

To predict important strategic issues in product safety during the next 10 years, the Health and Environmental Sciences Institute (HESI) of the International Life Sciences Institute initiated a mapping exercise to evaluate which issues are likely to be of societal, scientific, and regulatory importance to regulatory authorities, the HESI membership, and the scientific community at large. Scientists representing government, academia, and industry participated in the exercise. Societal issues identified include sensitive populations, alternative therapies, public education on the precautionary principle, obesity, and aging world populations. Scientific issues identified include cancer testing, children's health, mixtures and co-exposures, sensitive populations, idiosyncratic reactions, "omics" or bioinformatics, and environmental toxicology. Regulatory issues identified include national and regional legislation on chemical safety, exposure inputs, new technologies, transitioning new science into regulations and guidelines, conservative default factors, data quality, and sensitive populations. Because some issues were identified as important in all three areas (e.g. sensitive populations), a comprehensive approach to assessment and management is needed to ensure consideration of societal, scientific, and regulatory implications. The resulting HESI Combined Challenges Map is not intended to offer a universal description of challenges in safety assessment, nor is it intended to address, advocate, or manage the prioritized issues. Rather, the map focuses on and predicts issues likely to be central to the strategic agendas of individual companies and regulatory authorities in the developed world. Many of these issues will become increasingly important in the future in rapidly developing economies, such as India and China. The scientific mapping exercise has particular value to the toxicology community because it represents the contributions of key scientists from around the world from government, academia, and industry.

Dose response considerations in risk assessment--an overview of recent ILSI activities.

This paper will provide some perspective on the role that a consideration of the dose-response has played (past), is playing (present) and will play (future) in human risk assessment with special emphasis on a number of recent activities undertaken by various components of the International Life Sciences Institute (ILSI). The dose-response is a critically important concept in every aspect of biomedical science, including toxicology. A characterization of the dose response has been recognized as one of the four essential components of risk assessment since the release of the NRC/NAS report in 1983, and understanding the dose-response curve is the basis for regulatory toxicology. The introduction of concepts such as hormesis, thresholds of toxicological concern (TTC), and dose-dependent transitions in mechanisms of toxicity have emphasized the complexities associated with a characterization of the dose-response. The transitions to emphasizing predictive toxicology, systems biology, the new 'omics technologies, and high-throughput screening (HTS) have provided a new vision for toxicity testing. One impact of fully integrating these new concepts and technologies is that we will have unprecedented capabilities to explore the dose-response relationship, especially at low doses. How these new insights into the dose-response will affect our definition of threshold, and our understanding of the distinction between adverse and adaptive effects remain to be determined.

Relevance and follow-up of positive results in in vitro genetic toxicity assays: an ILSI-HESI initiative.

In vitro genotoxicity assays are often used to screen and predict whether chemicals might represent mutagenic and carcinogenic risks for humans. Recent discussions have focused on the high rate of positive results in in vitro tests, especially in those assays performed in mammalian cells that are not confirmed in vivo. Currently, there is no general consensus in the scientific community on the interpretation of the significance of positive results from the in vitro genotoxicity assays. To address this issue, the Health and Environmental Sciences Institute (HESI), held an international workshop in June 2006 to discuss the relevance and follow-up of positive results in in vitro genetic toxicity assays. The goals of the meeting were to examine ways to advance the scientific basis for the interpretation of positive findings in in vitro assays, to facilitate the development of follow-up testing strategies and to define criteria for determining the relevance to human health. The workshop identified specific needs in two general categories, i.e., improved testing and improved data interpretation and risk assessment. Recommendations to improve testing included: (1) re-examine the maximum level of cytotoxicity currently required for in vitro tests; (2) re-examine the upper limit concentration for in vitro mammalian studies; (3) develop improved testing strategies using current in vitro assays; (4) define criteria to guide selection of the appropriate follow-up in vivo studies; (5) develop new and more predictive in vitro and in vivo tests. Recommendations for improving interpretation and assessment included: (1) examine the suitability of applying the threshold of toxicological concern concepts to genotoxicity data; (2) develop a structured weight of evidence approach for assessing genotoxic/carcinogenic hazard; and (3) re-examine in vitro and in vivo correlations qualitatively and quantitatively. Conclusions from the workshop highlighted a willingness of scientists from various sectors to change and improve the current paradigm and move from a hazard identification approach to a "realistic" risk-based approach that incorporates information on mechanism of action, kinetics, and human exposure..

Framework for use of toxicity screening tools in context-based decision-making.

One of the principal applications of toxicology data is to inform risk assessments and support risk management decisions that are protective of human health. Ideally, a risk assessor would have available all of the relevant information on (a) the toxicity profile of the agent of interest; (b) its interactions with living systems; and (c) the known or projected exposure scenarios: to whom, how much, by which route(s), and how often. In practice, however, complete information is seldom available. Nonetheless, decisions still must be made. Screening-level assays and tools can provide support for many aspects of the risk assessment process, as long as the limitations of the tools are understood and to the extent that the added uncertainty the tools introduce into the process can be characterized and managed. Use of these tools for decision-making may be an end in itself for risk assessment and decision-making or a preliminary step to more extensive data collection and evaluation before assessments are undertaken or completed and risk management decisions made. This paper describes a framework for the application of screening tools for human health decision-making, although with some modest modification, it could be made applicable to environmental settings as well. The framework consists of problem formulation, development of a screening strategy based on an assessment of critical data needs, and a data analysis phase that employs weight-of-evidence criteria and uncertainty analyses, and leads to context-based decisions. Criteria for determining the appropriate screening tool(s) have been identified. The choice and use of the tool(s) will depend on the question and the level of uncertainty that may be appropriate for the context in which the decision is being made. The framework is iterative, in that users may refine the question(s) as they proceed. Several case studies illustrate how the framework may be used effectively to address specific questions for any endpoint of toxicity.

Immunotoxicogenomics: the potential of genomics technology in the immunotoxicity risk assessment process.

Evaluation of xenobiotic-induced changes in gene expression as a method to identify and classify potential toxicants is being pursued by industry and regulatory agencies worldwide. A workshop was held at the Research Triangle Park campus of the Environmental Protection Agency to discuss the current state-of-the-science of "immunotoxicogenomics" and to explore the potential role of genomics techniques for immunotoxicity testing. The genesis of the workshop was the current lack of widely accepted triggering criteria for Tier 1 immunotoxicity testing in the context of routine toxicity testing data, the realization that traditional screening methods would require an inordinate number of animals and are inadequate to handle the number of chemicals that may need to be screened (e.g., high production volume compounds) and the absence of an organized effort to address the state-of-the-science of toxicogenomics in the identification of immunotoxic compounds. The major focus of the meeting was on the theoretical and practical utility of genomics techniques to (1) replace or supplement current immunotoxicity screening procedures, (2) provide insight into potential modes or mechanisms of action, and (3) provide data suitable for immunotoxicity hazard identification or risk assessment. The latter goal is of considerable interest to a variety of stakeholders as a means to reduce animal use and to decrease the cost of conducting and interpreting standard toxicity tests. A number of data gaps were identified that included a lack of dose response and kinetic data for known immunotoxic compounds and a general lack of data correlating genomic alterations to functional changes observed in vivo. Participants concluded that a genomics approach to screen chemicals for immunotoxic potential or to generate data useful to risk assessors holds promise but that routine use of these methods is years in the future. However, recent progress in molecular immunology has made mode and mechanism of action studies much more practical. Furthermore, a variety of published immunotoxicity studies suggest that microarray analysis is already a practical means to explore pathway-level changes that lead to altered immune function. To help move the science of immunotoxicogenomics forward, a partnership of industry, academia, and government was suggested to address data gaps, validation, quality assurance, and protocol development.

Mode of action in relevance of rodent liver tumors to human cancer risk.

Hazard identification and risk assessment paradigms depend on the presumption of the similarity of rodents to humans, yet species specific responses, and the extrapolation of high-dose effects to low-dose exposures can affect the estimation of human risk from rodent data. As a consequence, a human relevance framework concept was developed by the International Programme on Chemical Safety (IPCS) and International Life Sciences Institute (ILSI) Risk Science Institute (RSI) with the central tenet being the identification of a mode of action (MOA). To perform a MOA analysis, the key biochemical, cellular, and molecular events need to first be established, and the temporal and dose-dependent concordance of each of the key events in the MOA can then be determined. The key events can be used to bridge species and dose for a given MOA. The next step in the MOA analysis is the assessment of biological plausibility for determining the relevance of the specified MOA in an animal model for human cancer risk based on kinetic and dynamic parameters. Using the framework approach, a MOA in animals could not be defined for metal overload. The MOA for phenobarbital (PB)-like P450 inducers was determined to be unlikely in humans after kinetic and dynamic factors were considered. In contrast, after these factors were considered with reference to estrogen, the conclusion was drawn that estrogen-induced tumors were plausible in humans. Finally, it was concluded that the induction of rodent liver tumors by porphyrogenic compounds followed a cytotoxic MOA, and that liver tumors formed as a result of sustained cytotoxicity and regenerative proliferation are considered relevant for evaluating human cancer risk if appropriate metabolism occurs in the animal models and in humans.

Assessing the potential to induce respiratory hypersensitivity.

Acute and repeat dose inhalation studies have been an important part of the safety assessment of drugs, chemicals, and other products throughout the world for many years. It is known that damage to the respiratory tract can be triggered either by nonspecific irritation or by specific immune-mediated pathogenesis, and it is acknowledged that traditional inhalation studies are not designed to address fully the impact of the latter. It is also recognized that different types of immune-mediated responses can be triggered by different classes of compounds and that some immune reactions in the lung are life threatening. As such, it is important to understand as fully as possible the basis for the immune-mediated damage to the lung in order to characterize adequately the risks of individual chemicals or proteins. It is against this background that a review of the methods used to assess the potential for immune-mediated respiratory hypersensitivity was conducted. The primary objectives of this review are to discuss appropriate methods for identifying and characterizing respiratory hypersensitivity hazards and risks; and to identify key data gaps and related research needs with respect to respiratory hypersensitivity testing. The following working definition of respiratory hypersensitivity was formulated: a hypersensitivity response in the respiratory tract precipitated by a specific immune response, mediated by multiple mechanisms, including IgE antibody. Because of the importance played by various classes of compounds, the subsequent sections of this review will consider protein-specific, chemical-specific, and drug-specific aspects of respiratory hypersensitivity.

In silico methods for evaluating human allergenicity to novel proteins: International Bioinformatics Workshop Meeting Report, 23-24 February 2005.

The ILSI Health and Environmental Sciences Institute (HESI) hosted an expert workshop 22-24 February 2005 in Mallorca, Spain, to review the state-of-the-science for conducting a sequence homology/bioinformatics evaluation in the context of a comprehensive allergenicity assessment for novel proteins, to obtain consensus on the value and role of bioinformatics in evaluating novel proteins, and to discuss the utility and methods of allergen-specific IgE testing in the diagnosis of food allergy. The workshop participants included over forty international experts from academia, industry, and government. The workshop was hosted by the HESI Protein Allergenicity Technical committee, which has established a long-term program whose mission is to advance the scientific understanding of the relevant parameters for characterizing the allergenic potential of novel proteins.

A proposed testing framework for developmental immunotoxicology (DIT).

A group of thirty immunotoxicology experts from the U.S. and E.U. representing government, industry, and academia met in May 2003, in Washington, D.C., to reach consensus regarding the most appropriate methods to assess developmental immunotoxicology (DIT) for hazard identification, including under what conditions such testing might be required. The following points represent the major conclusions from this roundtable discussion: (1) the rat is the preferred model; (2) any DIT protocol should be based on immune assays already validated; (3) DIT methods should be incorporated into standard developmental and reproductive toxicity protocols to the extent possible rather than a "stand-alone" protocol; (4) the approach to address DIT potential should be similar for chemicals and drugs, but the experimental design should be flexible and should reflect the specific questions to be answered; (5) it is possible to utilize a study design that assesses all critical windows in one protocol, with the results leading to further study of specific effects, as warranted; (6) animals should be exposed throughout the treatment protocol; (7) the triggers for DIT may include structure-activity-relationships, results from other toxicity studies, the intended use of a drug/chemical and/or its anticipated exposure of neonates and/or juveniles.

Developmental toxicology evaluations--issues with including neurotoxicology and immunotoxicology assessments in reproductive toxicology studies.

Developmental and reproductive toxicology (DART) has routinely been a part of safety assessment. Attention is now focused on the effects of chemicals on the developing nervous and immune systems. This focus on developmental neurotoxicology (DNT) and developmental immunotoxicology (DIT) is based on the premise that children differ from adults in some aspects of their biology and, thus, may also differ in their responses to chemicals. This session's objective was to discuss issues common to DNT and DIT as they relate to DART protocols, including high dose selection and maternal toxicity, adequacy of pup exposure during lactation, use of a different dosing paradigm for DART versus DNT or DIT studies, and whether DIT and DNT endpoints can be incorporated into a single DART study for hazard identification purposes. Consensus was achieved on all topics except the adequacy for risk assessment purposes of the use of a limited number of endpoints for DIT and DNT, with the DNT endpoints being the primary focus of disagreement. Panelists indicated that a combination study design for hazard identification was feasible, though flexibility to meet the scientific needs of the project was emphasized. The adequacy of existing triggers for additional developmental studies was also questioned. Panelists iterated the importance of understanding pup exposure during the various life stages and the use of toxicokinetic data in designing these studies. The group agreed to consider the HESI ACSA Life Stages Task Force recommendations as a next step to address some of the issues and challenges raised during this session.

Research strategies for safety evaluation of nanomaterials, part II: toxicological and safety evaluation of nanomaterials, current challenges and data needs.

This article summarizes a roundtable discussion held at the 2005 Society of Toxicology Annual Meeting in New Orleans, LA. The purpose of the roundtable was to review the current challenges and data needs for conducting toxicological and safety evaluations for nanomaterials, with the goals of presenting the current state-of-the science on the safety of nanomaterials and bringing together scientists representing government, academia, and industry to identify priorities for developing data to facilitate risk assessments for these materials. In this summary, the unique physicochemical properties associated with nanomaterials are reviewed in the context of the difficulties associated with measuring and characterizing them. In addition, the development of appropriate hazard data, the collection of accurate human and environmental exposure information, and the development of a better fundamental understanding of the modes of action for nanomaterials are discussed as factors that will impact the development of comprehensive toxicological and safety evaluations.

Workshop overview: approaches to the assessment of the allergenic potential of food from genetically modified crops.

There is a need to assess the safety of foods deriving from genetically modified (GM) crops, including the allergenic potential of novel gene products. Presently, there is no single in vitro or in vivo model that has been validated for the identification or characterization of potential food allergens. Instead, the evaluation focuses on risk factors such as source of the gene (i.e., allergenic vs. nonallergenic sources), physicochemical and genetic comparisons to known allergens, and exposure assessments. The purpose of this workshop was to gather together researchers working on various strategies for assessing protein allergenicity: (1) to describe the current state of knowledge and progress that has been made in the development and evaluation of appropriate testing strategies and (2) to identify critical issues that must now be addressed. This overview begins with a consideration of the current issues involved in assessing the allergenicity of GM foods. The second section presents information on in vitro models of digestibility, bioinformatics, and risk assessment in the context of clinical prevention and management of food allergy. Data on rodent models are presented in the next two sections. Finally, nonrodent models for assessing protein allergenicity are discussed. Collectively, these studies indicate that significant progress has been made in developing testing strategies. However, further efforts are needed to evaluate and validate the sensitivity, specificity, and reproducibility of many of these assays for determining the allergenicity potential of GM foods.

Developmental immunotoxicity testing: a review.

There are currently no validated or widely accepted methods for evaluating the effects of a chemical on the developing immune system. Nonetheless because of concerns over children's health issues, specifically the possibility that the very young are uniquely susceptible to chemical perturbation, governmental regulators are beginning to ask for information about potential effects on the developing immune system. This paper will address the following three goals. First, an update of the regulatory pressures for developmental immunotoxicology will be presented from a U.S. perspective. Second, an update on the state of the science of developmental immunotoxicology will be presented with an emphasis on results from a recent collaboration between Dow, DuPont and Cornell University. Finally, the principle conclusions from a recent ILSI/HESI workshop will be summarized. At this workshop, it was generally acknowledged that there are a variety of techniques available for assessing immunosuppression in adult animal models. However, it was emphasized that there is uncertainty about how to apply these approaches to a developing animal, especially if the goal is to have some standard procedure(s) that could be applied for regulatory risk assessment. Ultimately, the primary conclusion from this workshop was that developmental immunotoxicology, as a science, is still in its infancy and is not ready to be applied in a risk assessment strategy.

Autoimmunity by pesticides: a critical review of the state of the science.

The goals of this paper will be to present a critical review of the state of the science of pesticides and autoimmunity, and to discuss research that addresses the potential links between environmental chemicals and autoimmune disease. To date, the science of immunotoxicology has primarily focused on immunosuppression and hypersensitivity/allergy, and test methods are available to address these outcomes. So much progress has been made to address immunosuppression and contact sensitization that there are regulatory guidelines in the U.S. included in the registration of pesticides. In contrast, there are no validated approaches to assess autoimmunity. The overall objective of this paper will be to use pesticides as an important class of environmental chemicals to critically evaluate the state of the science for addressing chemical-induced autoimmunity. Specific examples of studies with pesticides will be discussed in the context of the following types of approaches: animal studies using standard immunotoxicological parameters; animal studies using specialized models of autoimmunity; human studies after environmental or occupational exposure; and human studies after accidental poisoning.

Skf 525-A induces cocaine N-demethylase, ethoxyresorufin O-deethylase, and pentoxyresorufin O-dealkylase activities by induction of cytochrome p-450 2B in female B6C3F1 mice.

Studies demonstrated that cocaine-induced immunosuppression is mediated by metabolites of cocaine. Although SKF 525-A inhibited cocaine N-demethylation in liver S9 fractions isolated from female B6C3F1 mice, our study showed that pretreatment of mice with SKF 525-A potentiated cocaine-induced suppression of the antibody response to sheep red blood cells. An increase in formaldehyde generation was subsequently shown following incubation of cocaine with the S9 fractions prepared from SKF 525-A-treated mice, indicating the possibility of cytochrome P-450 (CYP) induction. Therefore, the inductive effects of SKF 525-A on CYP enzyme activities and proteins were investigated in female B6C3F1 mice to elucidate the potentiation of cocaine-induced immunosuppression by SKF 525-A. When SKF 525-A was administered at 10, 20, or 40 mg/kg/d intraperitoneally for 7 consecutive days, both ethoxyresorufin O-deethylase and pentoxyresorufin O-dealkylase activities were induced dose-dependently. Furthermore, the induction of enzymatic activity was time dependent. Meanwhile, when the type of isozyme induced by SKF 525-A was analyzed by Western immunoblotting with monospecific anti-CYP 1A and anti-CYP 2B antibodies, only the CYP 2B appeared to be induced. From in vitro inhibition studies with monoclonal antibodies, it was confirmed that the induced activity of ethoxyresorufin O-deethylase by SKF 525-A was due to increased levels of CYP 2B proteins. Our present results provide an explanation for the potentiation of cocaine-induced immunosuppression by repeated exposure to SKF 525-A. Our results also indicate that ethoxyresorufin O-deethylase, a selective substrate for CYP 1A, may also be catalyzed by CYP 2B.

Preliminary study to test the feasibility of using a local lymph node type of approach to characterize respiratory allergens.

There is a critical need to develop animal models that can characterize the potential of respiratory allergy. Dust-mite allergens are one of the major etiological agents in the induction of allergy and asthma in humans. In this study, the effects of intratracheal injection with dust-mite allergen were investigated by analyzing the in vivo proliferative response of lung-draining hilar lymph nodes and histopathological changes in the lung parenchyma. Balb/c mice were inoculated intratracheally with dust-mite allergens, a mixture of Dermatophagoides farinae and D. pteronyssinus dissolved in phosphate-buffered saline, or with an equal volume of saline alone. After 1 week, all the mice were injected intravenously with radioactive (3)H-thymidine and sacrificed 5 h later so as to assess the radioactivity incorporated into the hilar lymph nodes. The results indicated a marked increase in the proliferative response in the hilar lymph nodes of the animals treated with the dust-mite allergen as compared to the response of the control group. Treatment with dust-mite allergen also caused perivascular and interstitial eosinophilic inflammation of the lungs, hyperplasia of bronchus-associated lymphoid tissue, and an increase in the eosinophil peroxidase activity in the lungs. These results indicate that intratracheal injection with dust-mite allergen can trigger a number of changes consistent with respiratory allergy, including an increased proliferation in the draining lymph nodes.

Lessons learned, challenges, and opportunities: the U.S. Endocrine Disruptor Screening Program.

In 1996, the U.S. Congress passed the Food Quality Protection Act and amended the Safe Drinking Water Act (SDWA) requiring the U.S. Environmental Protection Agency (EPA) to implement a screening program to investigate the potential of pesticide chemicals and drinking water contaminants to adversely affect endocrine pathways. Consequently, the EPA launched the Endocrine Disruptor Screening Program (EDSP) to develop and validate estrogen, androgen, and thyroid (EAT) pathway screening assays and to produce standardized and harmonized test guidelines for regulatory application. In 2009, the EPA issued the first set of test orders for EDSP screening and a total of 50 pesticide actives and 2 inert ingredients have been evaluated using the battery of EDSP Tier 1 screening assays (i.e., five in vitro assays and six in vivo assays). To provide a framework for retrospective analysis of the data generated and to collect the insight of multiple stakeholders involved in the testing, more than 240 scientists from government, industry, academia, and non-profit organizations recently participated in a workshop titled "Lessons Learned, Challenges, and Opportunities: The U.S. Endocrine Disruptor Screening Program." The workshop focused on the science and experience to date and was organized into three focal sessions: (a) Performance of the EDSP Tier 1 Screening Assays for Estrogen, Androgen, and Thyroid Pathways; (b) Practical Applications of Tier 1 Data; and (c) Indications and Opportunities for Future Endocrine Testing. A number of key learnings and recommendations related to future EDSP evaluations emanated from the collective sessions.