The ultimate goal of the Three Rs is the full replacement of animals used in biomedical research and testing. However, replacement is unlikely to occur in the near future; therefore the scientific community as a whole must continue to devote considerable effort to ensure optimal animal welfare for the benefit of the science and the animals, i.e., the R of refinement. Laws governing the care and use of laboratory animals have recently been revised in Europe and the US and these place greater emphasis on promoting the well-being of the animals in addition to minimizing pain and distress. Social housing for social species is now the default condition, which can present a challenge in certain experimental settings and for certain species. The practice of positive reinforcement training of laboratory animals, particularly non-human primates, is gathering momentum but is not yet universally employed. Enhanced consideration of refinement extends to rodents, particularly mice, whose use is still increasing as more genetically modified models are generated. The wastage of extraneous mice and the method of their euthanasia are refinement issues that still need to be addressed. An international, concerted effort into defining the needs of laboratory animals is still necessary to improve the quality of the animal models used as well as their welfare.

Despite wide-spread consensus on the need to transform toxicology and risk assessment in order to keep pace with technological and computational changes that have revolutionized the life sciences, there remains much work to be done to achieve the vision of toxicology based on a mechanistic foundation. A workshop was organized to explore one key aspect of this transformation – the development of Pathways of Toxicity (PoT) as a key tool for hazard identification based on systems biology. Several issues were discussed in depth in the workshop: The first was the challenge of formally defining the concept of a PoT as distinct from, but complementary to, other toxicological pathway concepts such as mode of action (MoA). The workshop came up with a preliminary definition of PoT as “A molecular definition of cellular processes shown to mediate adverse outcomes of toxicants”. It is further recognized that normal physiological pathways exist that maintain homeostasis and these, sufficiently perturbed, can become PoT. Second, the workshop sought to define the adequate public and commercial resources for PoT information, including data, visualization, analyses, tools, and use-cases, as well as the kinds of efforts that will be necessary to enable the creation of such a resource. Third, the workshop explored ways in which systems biology approaches could inform pathway annotation, and which resources are needed and available that can provide relevant PoT information to the diverse user communities.

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 findings and recommendations related to future EDSP evaluations emanated from the collective sessions.

Emerging challenges of managing and interpreting large amounts of complex biological data have given rise to the growing field of computational biology. We investigated the applicability of an integrated systems toxicology approach on five selected pesticides to get an overview of their modes of action in humans, to group them according to their modes of action, and to hypothesize on their potential effects on human health.
We extracted human proteins associated with prochloraz, tebuconazole, epoxiconazole, procymidone, and mancozeb and enriched each protein set by using a high confidence human protein interactome. Then we explored modes of action of the chemicals by integrating protein-disease information into the resulting protein networks. The dominating resulting human adverse effects were reproductive disorders followed by adrenal diseases.
Our results indicated that prochloraz, tebuconazole, and procymidone exert their effects mainly via interference with steroidogenesis and nuclear receptors. Prochloraz was associated with a large number of human diseases and, together with tebuconazole, showed several significant associations with testicular dysgenesis syndrome. Mancozeb showed a differential mode of action involving inflammatory processes.
This method provides an efficient way of overviewing data and grouping chemicals according to their mode of action and potential human adverse effects. Such information is valuable when dealing with predictions of mixture effects of chemicals and may contribute to the development of adverse outcome pathways.

The REACH (Registration, Evaluation, Authorization and restriction of Chemicals) and BPR (Biocidal Product Regulation) regulations strongly promote the use of non-animal testing techniques to evaluate chemical risk. This has renewed the interest towards alternative methods such as QSAR in the regulatory context. The assessment of bioconcentration factor (BCF) required by these regulations is expensive, in terms of costs, time, and laboratory animal sacrifices. Herein, we present QSAR models based on the ANTARES dataset, which is a large collection of known and verified experimental BCF data. Among the models developed, the best results were obtained from a nine-descriptor highly predictive model. This model was derived from a training set of 608 chemicals and challenged against a validation and blind set containing 152 and 76 chemicals, respectively. The model’s robustness was further controlled through several validation strategies and the implementation of a multi-step approach for the applicability domain. Suitable safety margins were used to increase sensitivity. The easy interpretability of the model is ensured by the use of meaningful biokinetics descriptors. The satisfactory predictive power for external compounds suggests that the new models could represent a reliable alternative to the in vivo assay, helping the registrants to fulfill regulatory requirements in compliance with the ethical and economic necessity to reduce animal testing.

Over the last 20 years, continuous advances in the field of molecular biology have led to the development of new strategies to discover and produce monoclonal antibodies, notably by phage display. Here we describe a simple procedure for antibody selection that considerably reduces the undesired selection of non-specific antibodies based on the use of biotinylated GST proteins fused to a target antigenic sequence. This procedure was tested on a collection of 7 different targets and resulted in the selection of a high percentage (71%) of antibodies specific for each target. This simple and effective in vitro procedure has great potential to replace animal immunization for the development of specific antibodies.

Early detection of cancer development is crucial for successful therapy and for monitoring patient outcome. Various immunodiagnostic methods are able to detect pathological changes in the human body ahead of symptomatic manifestation of the disease. Most immunological examinations are based on the detection of specific tumor markers in body fluids. Of the various cancer-specific proteins used for breast cancer diagnostics, one of the most commonly applied is the cancer antigen 15-3 (CA 15-3). An elevation in its serum level (>25-40 U/ml) usually correlates with tumor malignancy. The CA 15-3 antigen is also used for monitoring patients after surgical treatment and for measuring therapeutic efficacy. Herein, we present the generation of polyclonal IgY antibodies isolated from egg yolks of immunized hens and their application for CA 15-3 detection. The developed sandwich ELISA assay showed a detection limit of 0.028 U/ml, thus demonstrating its potential for clinical applications.

A special session at the Toxicology and Risk Assessment Conference in Cincinnati, OH, USA in May, 2012 presented approaches to expand current uses of in vitro toxicity data for risk assessment. Evaluation of xenobiotics through use of in vitro study methods is increasing exponentially and these methodologies offer a relatively fast and considerably cheaper way to determine toxicities in comparison to traditional animal-based approaches. One of the challenges with in vitro data is to effectively use this information for risk assessment purposes. Currently, in vitro studies are used as supportive information for hazard characterization and to identify mechanisms associated with toxicity. Being able to effectively correlate in vitro effects with in vivo observations represents a major challenge for risk assessors. The presentations in this special session provided innovative approaches toward effectively using in vitro data for the human health risk assessment process.