21st Century Approaches for Evaluating Exposures, Biological Activity, and Risks of Complex Substances: Workshop highlights.

The June 2019 workshop 21st Century Approaches for Evaluating Exposures, Biological Activity, and Risks of Complex Substances, co-organised by the International Council of Chemical Association's Long-Range Research Initiative and the European Commission's Joint Research Centre, is summarised. Focus was the need for improved approaches to evaluate the safety of complex substances. Approximately 10% and 20% of substances registered under the EU chemicals legislation are 'multi-constituent substances' and 'substances of unknown or variable compositions, complex reaction products and biological substances' (UVCBs), respectively, and UVCBs comprise approximately 25% of the U.S. Toxic Substances Control Act Inventory. Workshop participants were asked to consider how the full promise of new approach methodologies (NAMs) could be brought to bear to evaluate complex substances. Sessions focused on using NAMs for screening, biological profiling, and in complex risk evaluations; improving read-across approaches employing new data streams; and methods to evaluate exposure and dosimetry. The workshop concluded with facilitated discussions to explore actionable steps forward. Given the diversity of complex substances, no single 'correct' approach was seen as workable. The path forward should focus on 'learning by doing' by developing and openly sharing NAM-based fit-for-purpose case examples for evaluating biological activity, exposures and risks of complex substances.

Review of achievements of the OECD Working Party on Manufactured Nanomaterials' Testing and Assessment Programme. From exploratory testing to test guidelines.

This paper charts the almost ten years of history of OECD's work on nanosafety, during which the programme of the OECD on the Testing and Assessment of Manufactured Nanomaterials covered the testing of eleven nanomaterials for about 59 end-points addressing physical-chemical properties, mammalian and environmental toxicity, environmental fate and material safety. An overview of the materials tested, the test methods applied and the discussions regarding the applicability of the OECD test guidelines, which are recognised methods for regulatory testing of chemicals, are given. The results indicate that many existing OECD test guidelines are suitable for nanomaterials and consequently, hazard data collected using such guidelines will fall under OECD's system of Mutual Acceptance of Data (MAD) which is a legally binding instrument to facilitate the international acceptance of information for the regulatory safety assessment of chemicals. At the same time, some OECD test guidelines and guidance documents need to be adapted to address nanomaterials while new test guidelines and guidance documents may be needed to address endpoints that are more relevant to nanomaterials. This paper presents examples of areas where test guidelines or guidance for nanomaterials are under development.

Using default methodologies to derive an acceptable daily exposure (ADE).

This manuscript discusses the different historical and more recent default approaches that have been used to derive an acceptable daily exposure (ADE). While it is preferable to derive a health-based ADE based on a complete nonclinical and clinical data package, this is not always possible. For instance, for drug candidates in early development there may be no or limited nonclinical or clinical trial data. Alternative approaches that can support decision making with less complete data packages represent a variety of methods that rely on default assumptions or data inputs where chemical-specific data on health effects are lacking. A variety of default approaches are used including those based on certain toxicity estimates, a fraction of the therapeutic dose, cleaning-based limits, the threshold of toxicological concern (TTC), and application of hazard banding tools such as occupational exposure banding (OEB). Each of these default approaches is discussed in this manuscript, including their derivation, application, strengths, and limitations. In order to ensure patient safety when faced with toxicological and clinical data-gaps, default ADE methods should be purposefully as or more protective than ADEs derived from full data packages. Reliance on the subset of default approaches (e.g., TTC or OEB) that are based on toxicological data is preferred over other methods for establishing ADEs in early development while toxicology and clinical data are still being collected.

History of chronic toxicity and animal carcinogenicity studies for pharmaceuticals.

During the 20th century, as drug products were being developed to treat both known and emerging human diseases and conditions, determining the safety of these new chemicals became of increasing importance and necessity. For a time, the safety of use in human populations was of question, let alone whether the drug product was truly effective. As such, US and international regulatory agencies have played a major role in establishing standardized testing to evaluate the safety and efficacy of drug products. Pharmacologic and toxicologic evaluation of a new drug in animals is an important part of the pharmaceutical development process prior to its first-time use in humans, as well as its potential chronic use in affected populations. Just as both science and technology have evolved over the past century and further, so have the guidelines that have been put forth to adequately and efficiently evaluate the toxicity of new drugs and their subsequent safety in humans. This review summarizes the historical highlights of the conduct of drug safety evaluations in animals, particularly with regard to chronic toxicity and carcinogenicity assessments, and how we have progressed to our current standards and protocols to ensure safe use of drug products in human populations.

Toxicity testing in the 21st century: a vision and a strategy.

With the release of the landmark report Toxicity Testing in the 21st Century: A Vision and a Strategy, the U.S. National Academy of Sciences, in 2007, precipitated a major change in the way toxicity testing is conducted. It envisions increased efficiency in toxicity testing and decreased animal usage by transitioning from current expensive and lengthy in vivo testing with qualitative endpoints to in vitro toxicity pathway assays on human cells or cell lines using robotic high-throughput screening with mechanistic quantitative parameters. Risk assessment in the exposed human population would focus on avoiding significant perturbations in these toxicity pathways. Computational systems biology models would be implemented to determine the dose-response models of perturbations of pathway function. Extrapolation of in vitro results to in vivo human blood and tissue concentrations would be based on pharmacokinetic models for the given exposure condition. This practice would enhance human relevance of test results, and would cover several test agents, compared to traditional toxicological testing strategies. As all the tools that are necessary to implement the vision are currently available or in an advanced stage of development, the key prerequisites to achieving this paradigm shift are a commitment to change in the scientific community, which could be facilitated by a broad discussion of the vision, and obtaining necessary resources to enhance current knowledge of pathway perturbations and pathway assays in humans and to implement computational systems biology models. Implementation of these strategies would result in a new toxicity testing paradigm firmly based on human biology.

Dr. Daniel Acosta and In Vitro toxicology at the U.S. Food and Drug Administration's National Center for Toxicological Research.

For the past five years, Dr. Daniel Acosta has served as the Deputy Director of Research at the National Center for Toxicological Research (NCTR), a principle research laboratory of the U.S. Food and Drug Administration (FDA). Over his career at NCTR, Dr. Acosta has had a major impact on developing and promoting the use of in vitro assays in regulatory toxicity and product safety assessments. As Dr. Acosta nears his retirement we have dedicated this paper to his many accomplishments at the NCTR. Described within this paper are some of the in vitro studies that have been conducted under Dr. Acosta's leadership. These studies include toxicological assessments involving developmental effects, and the development and application of in vitro reproductive, heart, liver, neurological and airway cell and tissue models.

Personal reflections on Russell and Burch, FRAME, and the HSUS.

The coincidence of anniversaries associated with the publication of William Russell and Rex Burch's The Principles of Humane Experimental Technique, the founding of the Fund for the Replacement of Animals in Medical Experiments (FRAME), and the establishment of the collaboration between FRAME and the University of Nottingham, provides an opportunity to reflect on Russell and Burch's legacy and how it was carried forward by FRAME. The Principles, published in 1959, was the pioneering work in what later became the alternatives or Three Rs field of replacement, reduction, and refinement of animal use. Such was the book's initial and undeserved obscurity, however, that FRAME, following its founding in 1969, pioneered a similar approach independently of Russell and Burch's work. The Humane Society of the United States (HSUS) was also an early champion of the alternatives framework, and through the establishment of the Russell and Burch Award, helped unite Russell and Burch with what had emerged as the alternatives community. Thanks largely to FRAME, Russell and Burch were able to participate in Three Rs activity before their deaths. They lived long enough to see their ideas take hold, but not long enough to see the emerging revolution currently under way in toxicity testing, toward the use of non-animal methods.

The contribution of in vitro toxicity data in hazard and risk assessment: current limitations and future perspectives.

This paper discusses the role of in vitro toxicology in hazard and risk assessment. A short historical overview of the development of the field of in vitro toxicology is given, showing the possibilities, as well as the limitation for using in vitro methods in screening and in studying mechanisms of toxicity. Although the development of the use of in vitro methods over the last two or three decades is vast, the application in risk assessment is still limited. The need for a more integrated approach, i.e. using in vitro data not in isolation, but in combination with data on structure-activity and in silico biokinetic models is discussed. It is foreseen that the role of in vitro methods in future risk assessments will greatly enhance, also in the light of recent developments in technologies such as genomics and systems biology.

Challenges for Integrating Immunotoxicology into the Twenty-First-Century Toxicology Testing Paradigm.

An emerging emphasis on mechanism-focused and human-relevant alternatives to animal use in toxicology underlies the toxicology testing in the twenty-first-century initiative. Herein we describe in vitro high-throughput screening programs seeking to address this goal, as well as strategies established to integrate assay results to build weight of evidence in support of hazard assessment. Furthermore, we discuss unique challenges facing the application of such alternatives for assessing immunotoxicity given the complexity of immune responses. Addressing these challenges will require the development of novel in vitro assays that evaluate well-characterized biochemical processes involved in immune response to help inform on putative adverse outcomes in vivo.

Origin and evolution of high throughput screening.

This article reviews the origin and evolution of high throughput screening (HTS) through the experience of an individual pharmaceutical company, revealing some of the mysteries of the early stages of drug discovery to the wider pharmacology audience. HTS in this company (Pfizer, Groton, USA) had its origin in natural products screening in 1986, by substituting fermentation broths with dimethyl sulphoxide solutions of synthetic compounds, using 96-well plates and reduced assay volumes of 50-100 microl. A nominal 30 mM source compound concentration provided high microM assay concentrations. Starting at 800 compounds each week, the process reached a steady state of 7200 compounds per week by 1989. Screening in the Applied Biotechnology and Screening Group was centralized with screens operating in lock-step to maximize efficiency. Initial screens were full files run in triplicate. Autoradiography and image analysis were introduced for (125)I receptor ligand screens. Reverse transcriptase (RT) coupled with quantitative PCR and multiplexing addressed several targets in a single assay. By 1992 HTS produced 'hits' as starting matter for approximately 40% of the Discovery portfolio. In 1995, the HTS methodology was expanded to include ADMET targets. ADME targets required each compound to be physically detected leading to the development of automated high throughput LC-MS. In 1996, 90 compounds/week were screened in microsomal, protein binding and serum stability assays. Subsequently, the mutagenic Ames assay was adapted to a 96-well plate liquid assay and novel algorithms permitted automated image analysis of the micronucleus assay. By 1999 ADME HTS was fully integrated into the discovery cycle.

The Draize eye test.

Hundreds of substances are used daily that can damage eyesight. People's eyes are open to accidental or intentional exposure during the production, transportation, use, and disposal of chemical preparations. Ensuring the safety of consumer products was born during the mid twentieth century in the aftermath of chemical warfare research, and was motivated by the hazards of unsafe cosmetics. Justified by an exigency for public protection, the Draize eye test became a governmentally endorsed method to evaluate the safety of materials meant for use in or around the eyes. The test involves a standardized protocol for instilling agents onto the cornea and conjunctiva of laboratory animals. A sum of ordinal-scale items of the outer eye gives an index of ocular morbidity. Advances in ocular toxicology are challenging the validity, precision, relevance, and need of the Draize eye test. Preclinical product-safety tests with rabbits and other mammals also raise ethical concerns of animal wellbeing. Some use the Draize test as a rallying point for how animals are treated in science and industry. A battery of cellular systems and computer models aim to reduce and ultimately to replace whole-animal testing. Molecular measures of ocular toxicity may eventually allow comprehensive screening in humans. The Draize eye test was created and refined for humanitarian reasons and has assuredly prevented harm. Its destiny is to be progressively supplanted as in vitro and clinical alternatives emerge for assessing irritancy of the ocular surface.

A perspective on the progression of immunotoxicology.

Immunotoxicology originated in the early 1970s, when scientists began investigating chemicals. During the 1970s and early 1980s, a few investigators determined that chemicals were immunotoxic, developed and/or refined immunoassays, and began to characterize immunotoxic responses. In the 1980s, many new investigators entered the field, graduates were being trained as immunotoxicologists, the immune system was identified as a primary target organ, mechanisms of action studies proliferated, a comprehensive immunotoxicological panel was validated, the discipline gained universal credibility, and human studies emerged. The 1990s were ushered in with the concept of biological markers in immunotoxicology, a better understanding of "immune function", inclusion of immunotoxicology in risk assessment analysis, and a focus on molecular immunology. Future investigations will continue to improve and expand this foundation, pursue the relationship of immunotoxic chemicals and adverse health effects in humans, utilize genetically altered rodent models, and use gene expression technology to better understand the pathogenesis of immunotoxicological processes. Immunotoxicology has not only matured since its inception nearly 30 yr ago, but has become a prominent and respected discipline with global recognition; one that has made significant contributions to the advancement of the biomedical sciences.

Immunohistochemistry for light microscopy in safety evaluation of therapeutic agents: an overview.

The history of immunohistochemistry started in 1941 when Coons identified pneumococci using a direct fluorescent method. Then followed the indirect method, the addition of horseradish peroxidase, the peroxidase anti-peroxidase technique of 1979 and the use of the Avidin and Biotin complex in the early 1980s. This sequence of events can help one appreciate the differences in these various techniques and their increased sophistication and sensitivity. The technique has been applied in the field of safety evaluation of new pharmaceutical products. Examples of current projects are used to illustrate the scope of the application. The use of an antibody to detect proliferating cell nuclear antigen has, in a pilot study with the popliteal lymph node assay, provided a method of differentiating an irritant response to acetone from an immune response to hydrazine. In hydrazine-treated rats the proliferation is mainly in the follicular region whilst it is mainly sinusoidal in animals treated with acetone. In the guinea-pig maximisation test, initial work with dinitrochlorobenzene suggests that detection of Langerhans cells may aid the differentiation of an irritant from an immune response. The preclinical assessment of antibodies intended for therapeutic use in man requires immunohistochemistry to be used to identify any human tissues which show a cross-reactivity. The major problems are not in the test itself but in obtaining suitable material. Identification of hormones is a useful tool for assessing the effects of releasing factors and has proved useful in aiding tumour identification in routine carcinogenicity studies. In a rare case, detection of prolactin in cell deposits in the lungs of a rat confirmed that this was a metastasis from a pituitary carcinoma. The application of immunohistochemical techniques to preclinical assessment of drugs should always be considered. In particular, it is recommended that appropriate samples should be conserved from routine studies in order to permit these techniques to be performed, if considered appropriate in the light of findings during routine histological examination.

Acute systemic toxicity--prospects for tiered testing strategies.

After many years of controversy and debate, the LD50 test was finally deleted by the end of 2002. Three alternative animal tests, the Fixed Dose Procedure, the Acute Toxic Class Method and the Up and Down Procedure have been developed which give rise to significant improvements in animal welfare. They have recently undergone revision to improve their scientific performance but more importantly to increase their regulatory acceptance. They can now be used within a strategy for acute toxicity testing for all types of test substances and for all regulatory and in-house purposes. In vitro cytotoxicity tests could be used as adjuncts to these alternative animal tests within the next year or so to improve dose level selection and thus give further modest improvements in the numbers of animals used. However, the total replacement of animal tests requires a considerable amount of further test development, followed by validation, and is at least 10 years away.

Pathway-based toxicity: history, current approaches and liver fibrosis and steatosis as prototypes.

The Human Toxicology Project Consortium (HTPC) was created to accelerate implementation of the science and policies required to achieve a pathway-based foundation for toxicology as articulated in the 2007 National Research Council report, Toxicity Testing in the 21st Century: a Vision and a Strategy. The HTPC held a workshop, "Building Shared Experience to Advance Practical Application of Pathway-Based Toxicology: Liver Toxicity Mode-of-Action," in January, 2013, in Baltimore, MD, to further the science of pathway-based approaches to liver toxicity. This review was initiated as a thought-starter for this workshop and has since been updated to include insights from the workshop and other activities occurring in 2013. The report of the workshop has been published elsewhere in this journal (Willett et al., 2014).

Historical data analyses and scientific knowledge suggest complete removal of the abnormal toxicity test as a quality control test.

In the early 1900s, the abnormal toxicity test (ATT) was developed as an auxiliary means to ensure safe and consistent antiserum production. Today, the ATT is utilized as a quality control (QC) release test according to pharmacopoeial or other regulatory requirements. The study design has not been changed since around 1940. The evidence of abnormal toxicity testing as a prediction for harmful batches is highly questionable and lacks a scientific rationale. Numerous reviews of historical ATT results have revealed that no reliable conclusions can be drawn from this QC measure. Modern pharmaceutical manufacturers have thorough control of the manufacturing process and comply with good manufacturing practice rules. Contaminants are appropriately controlled by complying with the validated manufacturing processes and strict QC batch release confirming batch-to-batch consistency. Recognizing that product safety, efficacy, and stability can be ensured with strict QC measures, nowadays most regulatory authorities do not require the ATT for most product classes. In line with the replacement, reduction, and refinement (3Rs) initiative, the test requirement has been deleted from approximately 80 monographs of the European Pharmacopoeia and for the majority of product classes in the United States. For these reasons, it is recommended that the ATT should be consistently omitted world-wide and be removed from pharmacopoeias and other regulatory requirements.

The early research and development of ebselen.

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one; PZ-51, DR-3305), is an organoselenium compound with glutathione peroxidase (GPx)-like, thiol-dependent, hydroperoxide reducing activity. As an enzyme mimic for activity of the selenoenzyme GPx, this compound has proved to be highly useful in research on mechanisms in redox biology. Furthermore, the reactivity of ebselen with protein thiols has helped to identify novel, selective targets for inhibitory actions on several enzymes of importance in pharmacology and toxicology. Importantly, the selenium in ebselen is not released and thus is not bioavailable, ebselen metabolites being excreted in bile and urine. As a consequence, initial concerns about selenium toxicity, fortunately, were unfounded. Potential applications in medical settings have been explored, notably in brain ischemia and stroke. More recently, there has been a surge in interest as new medical applications have been taken into consideration. The first publication on the biochemical effects of ebselen appeared 30 years ago (Müller et al.), which prompted the authors to retrace the early development from their perspective. It is a fascinating example of fruitful interaction between research-oriented industry and academia.

Vinclozolin: a case study on the identification of endocrine active substances in the past and a future perspective.

In the late 1980s vinclozolin was tested for prenatal developmental toxicity in rats for registration purposes in USA. At 1000mg/kgbw, 95% of all fetuses were female upon visual inspection (ano-genital distance determination). Anti-androgenic effects (AA) were also noted in a subsequent 2-generation study. These findings triggered mechanistic investigations at BASF and at US-EPA. Results published by the latter were the starting point of the endocrine disruption (ED) discussion in the 1990s. AA effects of vinclozolin are mediated by two metabolites, which have an antagonistic effect on the androgen receptor. Currently, determination of ED has become a major end-point in toxicology testing and the US-EPA has set up an elaborated testing paradigm to fulfill this requirement. Future screening for ED can be improved making use of new technologies. ED modes of action can be determined by three alternative (3R) methods. Steroid synthesis in H295R cells (1), androgen-receptor binding in modified yeast (2) and metabolomics (3). Using vinclozolin as a case study, results indicate: (1) an effect on steroid synthesis in vitro, (2) an antagonistic effect on the androgen receptor and (3) that the metabolome profile of vinclozolin is similar to that of other receptor mediated anti-androgens (e.g. flutamide).

Extrapolation, uncertainty factors, and the precautionary principle.

This essay examines the relationship between the precautionary principle and uncertainty factors used by toxicologists to estimate acceptable exposure levels for toxic chemicals from animal experiments. It shows that the adoption of uncertainty factors in the United States in the 1950s can be understood by reference to the precautionary principle, but not by cost-benefit analysis because of a lack of relevant quantitative data at that time. In addition, it argues that uncertainty factors continue to be relevant to efforts to implement the precautionary principle and that the precautionary principle should not be restricted to cases involving unquantifiable hazards.