Setting the stage for next-generation risk assessment with non-animal approaches: the EU-ToxRisk project experience.

In 2016, the European Commission launched the EU-ToxRisk research project to develop and promote animal-free approaches in toxicology. The 36 partners of this consortium used in vitro and in silico methods in the context of case studies (CSs). These CSs included both compounds with a highly defined target (e.g. mitochondrial respiratory chain inhibitors) as well as compounds with poorly defined molecular initiation events (e.g. short-chain branched carboxylic acids). The initial project focus was on developing a science-based strategy for read-across (RAx) as an animal-free approach in chemical risk assessment. Moreover, seamless incorporation of new approach method (NAM) data into this process (= NAM-enhanced RAx) was explored. Here, the EU-ToxRisk consortium has collated its scientific and regulatory learnings from this particular project objective. For all CSs, a mechanistic hypothesis (in the form of an adverse outcome pathway) guided the safety evaluation. ADME data were generated from NAMs and used for comprehensive physiological-based kinetic modelling. Quality assurance and data management were optimized in parallel. Scientific and Regulatory Advisory Boards played a vital role in assessing the practical applicability of the new approaches. In a next step, external stakeholders evaluated the usefulness of NAMs in the context of RAx CSs for regulatory acceptance. For instance, the CSs were included in the OECD CS portfolio for the Integrated Approach to Testing and Assessment project. Feedback from regulators and other stakeholders was collected at several stages. Future chemical safety science projects can draw from this experience to implement systems toxicology-guided, animal-free next-generation risk assessment.

Hurdles and signposts on the road to virtual control groups-A case study illustrating the influence of anesthesia protocols on electrolyte levels in rats.

Introduction: Virtual Control Groups (VCGs) represent the concept of using historical control data from legacy animal studies to replace concurrent control group (CCG) animals. Based on the data curation and sharing activities of the Innovative Medicine Initiatives project eTRANSAFE (enhancing TRANSlational SAFEty Assessment through Integrative Knowledge Management) the ViCoG working group was established with the objectives of i) collecting suitable historical control data sets from preclinical toxicity studies, ii) evaluating statistical methodologies for building adequate and regulatory acceptable VCGs from historical control data, and iii) sharing those control-group data across multiple pharmaceutical companies. During the qualification process of VCGs a particular focus was put on the identification of hidden confounders in the data sets, which might impair the adequate matching of VCGs with the CCG. Methods: During our analyses we identified such a hidden confounder, namely, the choice of the anesthetic procedure used in animal experiments before blood withdrawal. Anesthesia using CO2 may elevate the levels of some electrolytes such as calcium in blood, while the use of isoflurane is known to lower these values. Identification of such hidden confounders is particularly important if the underlying experimental information (e.g., on the anesthetic procedure) is not routinely recorded in the standard raw data files, such as SEND (Standard for Exchange of Non-clinical Data). We therefore analyzed how the replacement of CCGs with VCGs would affect the reproducibility of treatment-related findings regarding electrolyte values (potassium, calcium, sodium, and phosphate). The analyses were performed using a legacy rat systemic toxicity study consisting of a control and three treatment groups conducted according to pertinent OECD guidelines. In the report of this study treatment-related hypercalcemia was reported. The rats in this study were anesthetized with isoflurane. Results: Replacing the CCGs with VCGs derived from studies comprising both anesthetics resulted in a shift of control electrolyte parameters. Instead of the originally reported hypercalcemia the use of VCG led to fallacious conclusions of no observed effect or hypocalcemia. Discussion: Our study highlights the importance of a rigorous statistical analysis including the detection and elimination of hidden confounders prior to the implementation of the VCG concept.

Morphological sex reversal upon short-term exposure to endocrine modulators in juvenile fathead minnow (Pimephales promelas).

Indications of effects on fish endocrine system have been noted when exposed to effluents of sewage treatment plants and subsequently in the receiving surface waters. For screening purposes, the concentration of vitellogenin (VTG) in plasma is employed to detect potential exposure of fish, to (anti-)estrogenic substances. However, little is known about the variability of VTG determinations and morphological endpoints (secondary sexual characteristics) in fish under exposure conditions employing compounds with hormonal activity other than estrogens. An in vivo test system was established to study the effects of methyltestosterone (MT, a potential model androgen) and fadrozole (F, an aromatase inhibitor) as well as the combination of MT and F on juvenile, sexually undifferentiated fathead minnows (Pimephales promelas). Fish were exposed to those compounds continuously in the (nominal) microg/l range (MT, 10, 50 and 100 microg/l; F, 25, 50, 100 microg/l; MT+F, 10 microg MT per l +50 microg F per l), for 14 days (MT+F) or 21 days (MT and F) using a flow-through system. The concentration of VTG and the expression of VTG mRNA was determined using whole body homogenates in an enzyme linked immunosorbant assay (ELISA) or reverse transcription-polymerase chain reaction (RT-PCR), respectively. Exposure to MT alone led to de novo mRNA expression as well as up to a four-fold increase of VTG. F had no effect on the VTG mRNA expression and VTG protein synthesis. The combination of MT and F had no effect on VTG concentrations, however, this produced a strong masculinisation of the juvenile fish, e.g. after 13 days of exposure 100% of the fish showed typical male sex characteristics, e.g. formation of nose tubercles and pigmentation of the dorsal fin. The above findings suggest that in fish MT may be aromatised to an estrogen. F, on the other hand, inhibits testosterone aromatisation. Consequently, the combination of MT and F strongly morphologically masculinised the juvenile fathead minnows. VTG detection at the mRNA and protein level is a sensitive parameter, however, it does not provide for any information regarding the baseline "estrogenicity" of a given parent compound.

The involvement of pro-inflammatory cytokines in nephrogenic systemic fibrosis - a mechanistic hypothesis based on preclinical results from a rat model treated with gadodiamide.

The evidence for the potential involvement of gadolinium-based contrast agents (GBCAs) in the pathomechanism of nephrogenic systemic fibrosis (NSF), a rare but serious disease occurring in patients with severe or end-stage renal failure, has grown due to recent epidemiological and preclinical research. Nevertheless there is still uncertainty with regard to the prevailing patho-physiological processes that may lead to NSF. To examine the potential mechanism of the fibrotic skin changes we applied a recently published rat model of NSF for investigations into serum markers for inflammation. For this purpose male Wistar rats were treated either once, three, or eight times with a daily intravenous injection of 2.5 mmol/kg gadodiamide, the drug substance of the magnetic resonance imaging (MRI) agent Omniscan. Clinical observations, hematology, clinical pathology, histopathology including electron microscopy and gadolinium (Gd) determination in serum, skin, femur and liver tissue, and a multiplexed analysis of 70 protein serum markers were performed. Gd was detectable in the skin, femur, and liver of the gadodiamide-treated rats 6h after the first administration. Macroscopic skin changes, appearing as reddening and early scab formation, were observed in one animal after the third daily administration and affected all animals after 8 daily administrations. Microscopy revealed dermal infiltrations after three administrations, progressing towards inflammatory lesions, ulcerations and crusts. Among the investigated serum marker panel 13 cytokines were significantly (p<0.01) elevated 6 h after the first injection, and eight stayed elevated over all time points: the monocyte chemotactic proteins MCP-1 and MCP-3, the macrophage inflammatory proteins MIP-1beta and MIP-2, the tumor necrosis factor TNF-alpha, the extracellular matrix regulator tissue inhibitor of metalloproteinase type 1 (TIMP-1), the vascular epithelial growth factor (VEGF) and osteopontin. The latter cytokine is of particular interest, since this matrix cellular glycoprotein is involved in the regulation of dystrophic calcification but also plays a role as a chemoattractant for dendritic cells, macrophages and T-lymphocytes, which in turn activate inflammatory pathways. Reflecting the physiological role of osteopontin, we hypothesize that Gd release from the GBCA-complex leads to the formation of insoluble Gd-deposits subsequently eliciting a physiological response similar to that seen during dystrophic calcification, i.e. an up-regulation of osteopontin and chemoattractant cytokines. Concomitant increase in vascular permeability caused by MIP-1, TNF-alpha and VEGF may lead to extravasation of chelated Gd or Gd-deposits. The inherent persistence of the Gd-deposits may subsequently result in an overactivation of pro-inflammatory pathways progressing towards overt skin effects.

Environmental risk assessment for the widely used iodinated X-ray contrast agent iopromide (Ultravist).

Iodinated X-ray contrast media are diagnostic pharmaceuticals that are applied to enhance the contrast between organs or vessels examined and surrounding tissues during radiography. These substances are applied in doses up to ca. 200 g per person (corresponding to approx 100 g iodine) and are rapidly excreted. In the sewage system they contribute to the burden of adsorbable organic halogens (AOX). To assess the potential environmental impact of this release, studies on environmental fate and effects were conducted for a risk assessment of the frequently used X-ray contrast medium iopromide (brand name: Ultravist). A screening test for biological degradation (OECD Screening Test 301 E) led to iopromide being classified as not readily biodegradable. Therefore, the predicted environmental concentration (PEC) in surface water was calculated in a first step. The resulting concentration of 2 microgram/liter was then compared in a second step with the predicted no-effect concentration as derived from a battery of ecotoxicity tests. In short-term toxicity tests with bacteria (Vibrio fisheri, Pseudomonas putida), algae (Scenedesmus subspicatus), crustaceans (Daphnia magna), and fish (Danio rerio, Leuciscus idus) no toxic effects were detected at the highest tested concentration of 10 g/liter. In a chronic toxicity test with D. magna no effect was observed at the highest tested concentration of 1 g/liter. Using an assessment factor of 100 the ratio between the predicted environmental concentration (PEC) and the predicted no-effect concentration (PNEC) was calculated to be

Biological degradation of cyclophosphamide and its occurrence in sewage water.

The mutagenic and cancerogenic antineoplastic agent cyclophosphamide (CP) is released into sewage water by cancer patient excretion. To assess the biological degradability of CP two standardized test systems, the Zahn-Wellens/EMPA test (OECD 302B) and a laboratory scale sewage treatment plant, were used. In both test systems the agent exhibited only poor degradability. To verify the expected occurrence of CP in hospital sewage, water samples were analyzed for CP with GC/MS after enrichment by solid-phase extraction. CP could be detected in concentrations ranging from 20 ng/L to 4.5 micrograms/L. The occurrence of the agent could also be proved in samples from the influent and the effluent of the communal sewage treatment plant into which the hospital's sewage water is shed. Concentrations ranged from 7 to 143 ng/L. In an attempt to assess the contribution of CP to the genotoxicity detected in hospital waste water in a recent study, the effects of CP in the umuC test, a bacterial genotoxicity assay, were investigated. However, no genotoxic effects of CP were found up to concentrations of 1 g/L.

[Tests of biodegradation of cytostatics cyclophosphamide and ifosfamide using the Closed Bottle Test (OECD 301 D)]. / Prüfung des biologischen Abbaus der Zytostatika Cyclophosphamid und Ifosfamid mit dem Closed Bottle Test (OECD 301 D).

Hospital and communal waste water differ with respect to their content of specific chemical substances like disinfectants or medicaments. Some medicaments are metabolized poorly by patients after administration. Antineoplastic substances are supposed to be carcinogen, mutagen and fetotoxic and embryotoxic substances. At the present there is no information available about their behaviour in the environment. Therefore the biodegradation of ifosfamide and cyclophosphamide was investigated by using the Closed Bottle Test (OECD 301 D). Both the structural isomeric antineoplastics were not biodegraded at a concentration of 5 mg/l in the Closed Bottle Test within 28 days ("not readily biodegradable"). A prolongation of the test up to 57 days did not alter the result. Abiotic elimination was neglectible. Cyclophosphamide and ifosfamide were not found toxic against waste water microorganisms. Further investigations about the elimination of ifosfamide and cyclophosphamide in the process of waste water treatment are necessary to get more knowledge about the possible risk connected with these substances.

Specific detection of membrane-toxic substances with a conductivity assay.

A conductivity assay that represents a new biotest able to detect the effects of membrane-toxic compounds, e.g., detergents, organic solvents, and radical formers, on various organisms was previously described and developed. The conductivity assay measures ion leakage from cells, tissues, or whole plant and animal organisms whose membrane systems have been damaged by membrane-toxic compounds. In this study the specificity of the conductivity assay for membrane-toxic compounds was tested by comparing the electrolyte efflux from Elodea canadensis leaves during incubation with a well-known detergent (benzalkonium chloride) using different plant physiological and biochemical techniques (photochemical efficiency, plasmolysis capacity, NBT reduction, and electron microscopy of membranes of E. canadensis leaves). The comparison of the different methods proved that the electrolyte loss during benzalkonium chloride incubation determined in the conductivity assay is due to membrane impairment. The observed electrolyte loss correlated with a reduction of photochemical efficiency and a decrease in both plasmolysis and NBT reduction capacity. Furthermore, a disintegration of the plasmalemma could be seen in the electron micrographs. These results indicate that the measured electrolyte loss in the conductivity assay is a specific effect of membrane-toxic compounds.