After the publication of the report titled Toxicity Testing in the 21st Century – A Vision and a Strategy, many initiatives started to foster a major paradigm shift for toxicity testing – from apical endpoints in animal-based tests to mechanistic endpoints through delineation of pathways of toxicity (PoT) in human cell based systems. The US EPA has funded an important project to develop new high throughput technologies based on human cell based in vitro technologies. These methods are currently being incorporated into the chemical risk assessment process. In the pharmaceutical industry, the efficacy and toxicity of new drugs are evaluated during preclinical investigations that include drug metabolism, pharmacokinetics, pharmacodynamics and safety toxicology studies. The results of these studies are analyzed and extrapolated to predict efficacy and potential adverse effects in humans. However, due to the high failure rate of drugs during the clinical phases, a new approach for a more predictive assessment of drugs both in terms of efficacy and adverse effects is getting urgent. The food industry faces the challenge of assessing novel foods and food ingredients for the general population, while using animal safety testing for extrapolation purposes is often of limited relevance. The question is whether the latest paradigm shift proposed by the Tox21c report for chemicals may provide a useful tool to improve the risk assessment approach also for drugs and food ingredients.

Intracranial lymphocytic choriomeningitis virus (LCMV) infection is a widely used animal model to study virus-induced cytotoxic T cell (CTL) mediated meningitis and immunopathology. This model causes severe pain and distress in mice, especially at later stages of the disease. Therefore, new treatment regimens to improve animal welfare have to be developed. In this study, we subcutaneously implanted ALZET® osmotic pumps continuously releasing buprenorphine to reduce pain in mice with LCMV-induced meningitis. Mice treated with buprenorphine demonstrated strongly reduced symptoms of pain. The LCMV-specific cytotoxic T cell response and the immune cell infiltration into the central nervous system (CNS) were unchanged in analgesia treated mice, indicating that the LCMV-induced immune response was not altered in these mice. Taken together, we demonstrate that in this animal model for meningitis continuous buprenorphine treatment improves animal welfare without affecting the immune response.

A simple and rapid luminometric assay for the detection of chemical inhibitors of human thyroid peroxidase (hTPO) activity was developed and validated with 10 model compounds. hTPO was derived from the human thyroid follicular cell line Nthy-ori 3-1 and its activity was quantified by measuring the oxidation of luminol in the presence of hydrogen peroxide (H2O2), which results in emission of light at 428 nm. In this assay, hTPO activity was shown to be inhibited by 5 known TPO inhibitors and not inhibited by 5 non-inhibitors. Similar results were obtained with porcine TPO (pTPO). The inhibition of hTPO by the model compounds was also tested with guaiacol and Ampliflu Red as alternative indicator substrates. While all substrates allowed the detection of pTPO activity and its inhibition, only the Ampliflu Red and luminol-based methods were sensitive enough to allow the quantification of hTPO activity from Nthy-ori 3-1 cell lysates. Moreover, luminol gave results with a narrower 95% confidence interval and therefore more reliable data. Whole extracts of fast-growing Nthy-ori 3-1 cells circumvent the need for animal-derived thyroid organs, thereby reducing costs, eliminating potential contamination and providing the possibility to study human instead of porcine TPO. Overall, the application of luminol and Nthy-ori 3-1 cell lysate for the detection of the disruption of hTPO activity was found to represent a valuable in vitro alternative and a possible candidate for inclusion within a high throughput integrated testing strategy for the detection of compounds that potentially interfere with normal thyroid function in vivo.

The tuberculin skin test is the method of choice for tuberculosis surveillance in livestock ruminants. The exact definition of the biological activity of bovine tuberculin purified protein derivatives (bovine tuberculin PPDs) is essential for the reliability of a test system. PPDs consist of heterogeneous mixtures of mycobacterial antigens, making it difficult to determine their potency in vitro. The commonly used batch potency test is therefore based on the evaluation of skin reactions in mycobacteria-sensitized guinea pigs. The aim of the present study was to test an alternative in vitro method that reliably quantifies tuberculin PPD potency. This novel approach may prevent animal distress in the future.
To this end a flow cytometry-based lymphocyte proliferation assay using peripheral blood mononuclear cells (PBMCs) from sensitized guinea pigs was established. Potency estimates for individual PPD preparations were calculated in comparison to an international standard. The comparison with results obtained from the guinea pig skin test revealed that the lymphocyte proliferation assay is more precise but results in systematically higher potency estimates. However, with a manufacturer specific correction factor a correlation of over 85% was achieved, highlighting the potential of this in vitro method to replace the current guinea pig skin test.

In April 2013 the mouse antibody serum neutralization test (SNT) was formally incorporated into European Pharmacopoeia monograph 0451 for potency testing of inactivated veterinary rabies vaccines. The SNT is designed to replace the highly variable and pain and distress causing NIH mouse rabies challenge assay. The adoption of the SNT meets the European ambition (i.e., EC and CoE) to replace, reduce and/or refine laboratory animal testing. However, regulatory acceptance and use of 3R models, such as the SNT, remains challenging. This paper aims at clarifying the process of acceptance and use of the SNT. For this purpose it reconstructs the process and reveals barriers and drivers that have been observed by involved stakeholders to have played a role. In addition it extracts lessons to stimulate regulatory acceptance in similar future processes. The incorporation of the SNT into the monographs went relatively quickly due to a thorough test development and pre-validation phase, commitment and cooperation of relevant stakeholders and a strong project coordination of the international validation study. The test was developed by the Paul Ehrlich Institut, a leading European OMCL. This facilitated its European regulatory use. The use by industry is in a critical phase. At this stage product specific validation and the question whether the SNT will be accepted outside Europe are important influencing factors.

Erratum: doi:10.14573/altex.1507131

Fluorescence proteins have been useful as genetic reporters for a wide range of applications in biomedical research and are frequently used for the analysis of transgene activity.
Here, we show that expression levels of the ubiquitously expressed fluorescent proteins eGFP, mCherry and tdTomato can be measured in transgenic mouse lines with random or targeted integrations. We identified the tail of the mouse as the tissue best suited for quantifying fluorescence intensity and show that expression levels in the tail correlate with gene dose. This allows for instant non-invasive determination of the genetic condition at the transgenic locus (hemizygous/heterozygous and homozygous) while simultaneously providing an objective comparison for transgene expression levels among different mouse lines.
In summary, we demonstrate for the first time that the gene dose of a ubiquitously expressed fluorescence reporter can be reliably quantified and directly linked to the genotype of transgenic mice. Based on this information, animals with the appropriate genotype can be instantly selected without laborious analysis for establishing and breeding of new transgenic lines, reducing the number of “waste” animals. Furthermore, no tissue sampling is necessary, which is a significant refinement of genotyping procedures. Both aspects are important improvements for the genotyping of transgenic mice that follow the principles of the 3Rs (reduction and refinement).