Michael P. Holsapple

t4 workshop report: 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.

What's so special about the developing immune system?

The evolution of the subdiscipline of developmental immunotoxicology (DIT) as it exists today has been shaped by significant regulatory pressures as well as key scientific advances. This review considers the role played by legislation to protect children’s health, and on the emergence of immunotoxcity and developmental immunotoxicity guidelines, as well as providing some context to the need for special attention on DIT by considering the evidence that the developing immune system may have unique susceptibilities when compared to the adult immune system. Understanding the full extent of this potential has been complicated by a paucity of data detailing the development of the immune system during critical life stages as well as by the complexities of comparisons across species. Notably, there are differences between humans and nonhuman species used in toxicity testing that include specific differences relative to the timing of the development of the immune system as well as more general anatomic differences, and these differences must be factored into the interpretation of DIT studies. Likewise, understanding how the timing of the immune development impacts on various immune parameters is critical to the design of DIT studies, parameters most extensively characterized to date in young adult animals. Other factors important to DIT, which are considered in this review, are the recognition that effects other than suppression (e.g., allergy and autoimmunity) are important; the need to improve our understanding of how to assess the potential for DIT in humans; and the role that pathology has played in DIT studies in test animals. The latter point receives special emphasis in this review because pathology evaluations have been a major component of standard nonclinical toxicology studies, and could serve an important role in studies to evaluate DIT. This possibility is very consistent with recommendations to incorporate a DIT evaluation into standard developmental and reproductive toxicology (DART) protocols. The overall objective of this review is to provide a ‘snapshot’ of the current state-of-the-science of DIT. Despite significant progress, DIT is still evolving and it is our hope that this review will advance the science.

Effects of N-nitrosodimethylamine on cell-mediated immunity.

Dimethylnitrosamine (DMN) exposure altered the cell‐mediated immune response of B6C3F1 adult female mice as assessed by several immunological assays. Following 14 daily exposures (i.p.) to 1.5, 3.0, or 5.0 mg DMN/kg, mice exhibited a depression in their lymphoproliferative response to the T‐cell mitogens concanavalin A and phytohemagglutinin, and in their mixed lymphocyte response to mitomycin‐treated DBA‐2 spleen cells. The delayed hypersensitivity response (DHR) to keyhole limpet hemocyanin (KLH), as measured by vascular permeability changes, was decreased by over 50% at the 5.0‐mg/kg dose. When the DHR to KLH was measured by an influx of endogenously 125l‐ iododeoxyuridine (lUdR)‐labelled monocytes, there was a 300% increase in the response of the 5.0‐mg‐DMN/kg group. Adoptive transfer studies using exogenously radiolabeled (51Cr) bone marrow cells from either vehicle‐ or DMN‐treated (5 mg/kg) donors indicated a >60% reduction in the DHR to KLH in DMN‐treated mice (5.0 mg/kg level) regardless of the donor treatment. Animals exposed to DMN exhibited a decreased susceptibility to Listeria monocytogenes. The dichotomy in the results of the KLH DHR measured by monocyte influx and the increased resistance to the bacterial challenge were interpreted to reflect an effect on bone marrow. The numbers of granulocyte/ monocyte stem cells were increased in a dose‐related fashion in bone marrow from DMN‐treated mice. The results indicate that DMN‐treatment impairs cell‐ mediated immunity while increasing the number of bone marrow cells differentiating to form granulocytes or monocytes with an apparent enhancement in functional activity.

Evidence-based toxicology for the 21st century: Opportunities and challenges

The Evidence-based Toxicology Collaboration (EBTC) was established recently to translate evidence-based approaches from medicine and health care to toxicology in an organized and sustained effort. The EBTC held a workshop on Evidence-based Toxicology for the 21st Century: Opportunities and Challenges in Research Triangle Park, North Carolina, USA on January 24-25, 2012. The presentations largely reflected two EBTC priorities: to apply evidence-based methods to assessing the performance of emerging pathway-based testing methods consistent with the 2007 National Research Council report on Toxicity Testing in the 21st Century as well as to adopt a governance structure and work processes to move that effort forward. The workshop served to clarify evidence-based approaches and to provide food for thought on substantive and administrative activities for the EBTC. Priority activities include conducting pilot studies to demonstrate the value of evidence-based approaches to toxicology, as well as conducting educational outreach on these approaches.

Accelerating the Development of 21st-Century Toxicology: Outcome of a Human Toxicology Project Consortium Workshop

The U.S. National Research Council (NRC) report on “Toxicity Testing in the 21st century” calls for a fundamental shift in the way that chemicals are tested for human health effects and evaluated in risk assessments. The new approach would move toward in vitro methods, typically using human cells in a high-throughput context. The in vitro methods would be designed to detect significant perturbations to “toxicity pathways,” i.e., key biological pathways that, when sufficiently perturbed, lead to adverse health outcomes. To explore progress on the report’s implementation, the Human Toxicology Project Consortium hosted a workshop on 9–10 November 2010 in Washington, DC. The Consortium is a coalition of several corporations, a research institute, and a non-governmental organization dedicated to accelerating the implementation of 21st-century Toxicology as aligned with the NRC vision. The goal of the workshop was to identify practical and scientific ways to accelerate implementation of the NRC vision. The workshop format consisted of plenary presentations, breakout group discussions, and concluding commentaries. The program faculty was drawn from industry, academia, government, and public interest organizations. Most presentations summarized ongoing efforts to modernize toxicology testing and approaches, each with some overlap with the NRC vision. In light of these efforts, the workshop identified recommendations for accelerating implementation of the NRC vision, including greater strategic coordination and planning across projects (facilitated by a steering group), the development of projects that test the proof of concept for implementation of the NRC vision, and greater outreach and communication across stakeholder communities.

Naphthalene Toxicity in CD-1 Mice: General Toxicology and Immunotoxicology

Random bred CD-1 mice were used to evaluate the acute oral toxicity and subchronic toxicity of naphthalene administered in corn oil. The acute oral LD50 of naphthalene was 533 and 710 mg/kg in male and female mice, respectively. Subchronic toxicity was evaluated with 14- and 90-day daily oral gavage studies. Doses utilized in the 14-day study were 27, 53, and 267 mg/kg, with the latter representing one-half of the male LD50. Both males and females demonstrated a 5-10% mortality and depressed body weight at the high dose only. Males had decreased thymus weights, and females had decreased spleen and increased lung weights at the high dose only. Other organ weights were unaffected at any dosage level. Serum enzyme and electrolyte levels were not altered in a dose-related manner. To assess the potential immunotoxicity of naphthalene the following screen was utilized: humoral immune response, response to mitogens, delayed hypersensitivity response, popliteal lymph node response, bone marrow stem cell number, and DNA synthesis. No evidence of immunotoxicity was demonstrated. The 90-day study employed daily oral doses of 5.3, 53, and 133 mg/kg. There was no treatment-related mortality in either sex, nor was body weight affected. Organ weights were not affected in males, and females showed reduced spleen weights only at the high dose. Serum enzyme and electrolyte levels, as well as the immunotoxicity screen, indicated that naphthalene doses up to one-fourth the LD50 for 90 days failed to elicit consistent statistically significant and biologically relevant compound-related effects. A screen of the effects of the 90-day naphthalene treatment on various aspects of the hepatic drug metabolizing system indicated no alterations, with the exception of a specific dose-related inhibition of aryl hydrocarbon hydroxylase activity in both male and female mice.

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.

The new revolution in toxicology: the good, the bad, and the ugly.

In 2007, the United States National Academy of Sciences issued a report entitled Toxicity Testing in the 21st Century: A Vision and a Strategy. The report reviewed the state of the science and outlined a strategy for the future of toxicity testing. One of the more significant components of the vision established by the report was an emphasis on toxicity testing in human rather than animal systems. In the context of drug development, it is critical that the tools used to accomplish this strategy are maximally capable of evaluating human risk. Since 2007, many advances toward implementation of this vision have been achieved, particularly with regard to safety assessment of new chemical entities intended for pharmaceutical use.

Strategies for the follow-up of positive results in the in vitro genotoxicity assays--an international collaborative initiative.

Identification of potential mutagens plays a critical role in the overall safety assessment of chemicals and pharmaceuticals because gene and chromosomal mutations can potentially lead to adverse health consequences, including cancer, reproductive impairment, developmental anomalies, or genetic diseases. Currently, a battery of tests is used to screen for the potential of an agent to induce mutations or chromosomal damage. This battery includes tests conducted in bacteria (popularly known as the Ames test), in cultured mammalian cells, and in rodents. While these tests have been generally effective in preventing the introduction of potent genotoxic agents into the market place, experience of the past couple of decades has also revealed that the in vitro mammalian cell culture-based assays for clastogenicity/mutagenicity generally yield a high rate of positive results. Importantly, a large number of the mammalian in vitro positive findings have not been confirmed in in vivo genotoxicity tests and have shown to be poor predictors of potential carcinogenicity in rodent bioassays. These findings raised significant concerns regarding the specificity of these assays and the relevance of the results in human risk assessment. In addition, positive in vitro results generally lead to further follow-up studies that are resource intensive including unnecessary use of animals. Furthermore, such positive in vitro results often lead to prohibiting the use and/or development of compounds of significant societal value despite the availability of other ancillary toxicity data indicating negligible concern for adverse human effects. Genetic toxicologists have recognized that there are still ample opportunities to improve and enhance the approaches used in testing, including the type of assays used, the follow-up strategies employed to resolve the relevance of positive findings, and the strategies used for data interpretation. Taking all of these points into consideration, scientists from industry, academia, and the government representing North America, Europe, and Japan initiated a project in 2006 under the auspices of the Health and Environmental Sciences Institute (HESI), a global branch of the International Life Sciences Institute (ILSI). This initiative titled ‘‘Relevance and Follow-up of Positive Results from In Vitro Genetic Toxicity’’ (IVGT) had the following key objectives:

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.