Application Of Test Substances Research Articles

A microphysiological system for parallelized morphological and electrophysiological read-out of 3D neuronal cell culture.

Three-dimensional in vitro models in microfluidic systems are promising tools for studying cell biology, with complex models using multiple cell types combined with high resolution imaging. Neuronal models demand electrical readout of the activity of networks of single neurons, yet classical planar microelectrode arrays struggle to capture extracellular action potentials when neural soma are suspended distant from the microelectrodes. This study introduces sophisticated microfluidic microelectrode arrays, specifically tailored for electrophysiology of 3D neuronal cultures. Using multilayer photolithography of permanent epoxy photoresists, we developed devices having 12 independent culture modules in a convenient format. Each module has two adjacent compartments for hydrogel-based 3D cell culture, with tunnels allowing projection of neurites between compartments. Microelectrodes integrated in the tunnels record action potentials as they pass between the compartments. Mesh ceilings separate the compartments from overlying wells, allowing for simple cell seeding and later nutrient, gas and waste exchange and application of test substances. Using these devices, we have demonstrated 3D neuronal culture, including electrophysiological recording and live imaging. This microphysiological platform will enable high-throughput investigation of neuronal networks for investigation of neurological disorders, neural pharmacology and basic neuroscience. Further models could include cocultures representing multiple brain regions or innervation models of other organs.

Results of extended plant tests using more realistic exposure scenarios for improving environmental risk assessment of veterinary pharmaceuticals.

BackgroundResidues of veterinary medicinal products (VMPs) enter the environment via application of manure onto agricultural areas where in particular antibiotics can cause phytotoxicity. Terrestrial plant tests according to OECD guideline 208 are part of the environmental risk assessment of VMPs. However, this standard approach might not be appropriate for VMPs which form non-extractable residues or transformation products in manure and manure-amended soil. Therefore, a new test design with a more realistic exposure scenario via manure application is needed. This paper presents an extended plant test and its experimental verification with the veterinary antibiotics florfenicol and tylosin tartrate. With each substance, plant tests with four different types of application were conducted: standard tests according to OECD 208 and three tests with application of test substance via spiked manure either without storage, aerobically incubated, or anaerobically incubated for different time periods.ResultsIn standard tests, the lowest NOEC was <0.06 mg/kg dry soil for florfenicol and 16.0 mg/kg dry soil for tylosin tartrate. Pre-tests showed that plant growth was not impaired at 22-g fresh manure/kg dry soil, which therefore was used for the final tests. The application of the test substances via freshly spiked as well as via aerobically incubated manure had no significant influence on the test results. Application of florfenicol via anaerobically incubated manure increased the EC10 by a factor up to 282 and 540 for half-maximum and for maximum incubation period, respectively. For tylosin tartrate, this factor amounted to 64 at half-maximum and 61 at maximum incubation period. The reduction of phytotoxicity was generally stronger when using cattle manure than pig manure and particularly in tests with cattle manure phytotoxicity decreased over the incubation period.ConclusionsThe verification of the extended plant test showed that seedling emergence and growth are comparable to a standard OECD 208 test and reliable effect concentrations could be established. As demonstrated in the present study, phytotoxicity of veterinary antibiotics can be significantly reduced by application via incubated manure compared to the standard plant test. Overall, the presented test design proved suitable for inclusion into the plant test strategy for VMPs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12302-016-0089-2) contains supplementary material, which is available to authorized users.

Dermal toxicity, eye and dermal irritation and skin sensitization evaluation of a new formulation of Bacillus thuringiensis var israelensis SH-14

Bacillus thuringiensis (Bt) is the best known and most widely used of all pesticidal microbes. The aim of this study was to assess the toxicity of a new formulation of Bacillus thuringiensis var israelensis SH-14 in rats through acute dermal toxicity, dermal and eye irritation experiments. The acute dermal toxicity and dermal and eye irritation studies were performed using rabbits according to the United States Environmental Protection Agency guidelines 885.3100, 870.2500 and 870.2500, respectively. The skin sensitization study was carried out in accordance to the EPA OPPTS 870.2600 using guinea pigs. There was no mortality and no evidence of treatment-related toxicity in acute dermal toxicity test. No dermal responses, including erythema/eschar or edema, were found in rabbits treated with the new formulation of Bti SH-14. Minimum response was observed after eye application of test substance. No skin sensitization reactions were observed after the challenge with the new formulation of Bti SH-14 in the Bti SH-14-treated guinea pigs. In summary, the present study demonstrated that the new formulation of Bti SH-14 is not acutely toxic via dermal route, has low eye irritation and would not cause dermal irritation or hypersensitivity to tested animals.

Raloxifene supports early fracture healing more than estrogen in ovariectomized rats

Summary Objectives: Most people suffering from osteoporosis are undiagnosed: the first osteopenic fracture strikes an untreated organism. Therefore implant fixation often fails and bone healing is disturbed. In this basic research project, we explore possibilities to improve the quality of the osteopenic bone immediately after a fracture to avoid these complications. Methods: Thirty-six female rats, which developed osteopenia within ten weeks duration after ovariectomy (OVX), underwent a standardized metaphyseal osteotomy with a bridging T-plate-fixation. After that rats were divided into three groups, which received soyfree food supplemented with raloxifene (R: 2.02 mg/d) or estradiol-17β-benzoate (E: 0.09 mg/d), orally, or soyfree food (SF) only. During fracture healing the rats were subcutaneously injected with fluorescent agents to help label and visualize the process of bone formation. Bones were analyzed using a three-point bending test, histological sections and microradiographs. Results: Raloxifene and estradiol have exerted anabolic effects on the trabecular bone. Both substances induced fracture healing mainly via endosteal callus formation (R: 2.08 ± 0.66 mm2, E: 2.02 ± 0.75 mm2 vs. SF: 1.78 ± 0.74 mm2). Due to early bridging and advanced fracture healing, less bone occurred in the later stages after application of test substances. The biomechanical features of the callus formations determined by the Yield load of R- (100.3 ± 28.4 N) were at the level of E-treated bone (93.8 ± 29.7 N) being higher in both comparing to the osteopenic bones (SF: 76.4 ± 18.8 N). Conclusions: Raloxifene and estrogen had supporting effects in the therapy of fractures of osteopenic bone. They improved not only the bone, but also the callus structure. The raloxifene- and estradiol-treatment enhanced the mechanical properties of the osteopenic bone, which probably lead to (micro) fracture risk reduction. Taking into account all the results there is an advantage for raloxifene; additionally its negative side effects detected in human settings, with respect to e. g. breast cancer propagation, are less than that of estrogens.

The isolated chicken eye test as a suitable in vitro method for determining the eye irritation potential of household cleaning products

Eye irritation is an important endpoint in the safety evaluation of consumer products and their ingredients. Several in vitro methods have been developed and are used by different industry sectors to assess eye irritation. One such in vitro method in use for some time already is the isolated chicken eye test (ICE). This investigation focuses on assessing the ICE as a method to determine the eye irritation potential of household cleaning products, both for product safety assurance prior to marketing and for classification and labeling decisions. The ICE involves a single application of test substances onto the cornea of isolated chicken eyes. Endpoints are corneal swelling, corneal opacity and fluorescein retention. The ICE results were compared to historic LVET data in this study due to availability of such in vivo data and the ability to correlate LVET to human experience data on the outcome of accidental exposures to household cleaning products in general. The results of this study indicate that the ICE test is a useful in vitro method for evaluating the eye irritation/corrosion potential and establishing classification and labeling for household cleaning products. For new product formulations, it is best used as part of a weight-of-evidence approach and benchmarked against data from comparable formulations with known eye irritation/corrosion profiles and market experience.

Prevention of compartment syndrome in dorsal root ganglia caused by exposure to nucleus pulposus.

An experimental study to clarify the effects of pentoxifylline, as an anti-tumor necrosis factor-alpha therapy on endoneurial fluid pressure in the dorsal root ganglion using an animal model of herniated nucleus pulposus. To investigate the effects of anti-tumor necrosis factor-alpha therapy to nucleus pulposus-induced nerve root/dorsal root ganglion changes. It has been reported experimentally that application of nucleus pulposus into epidural space induces morphologic and functional changes in the nerve roots and induces compartment syndrome in the dorsal root ganglia. Tumor necrosis factor-alpha has been considered a key pathogenic factor in the initiation and maintenance of neuropathic pain states. A total of 11 adult, female Sprague-Dawley rats had their left L5 nerve roots and associated dorsal root ganglions exposed. Autologous nucleus pulposus was applied to the L5 nerve root just proximal to the dorsal root ganglion. A piece of Spongel (Yamanouchi Pharmaceutical Co., Tokyo) containing 20 microL of 1000 microg/mL pentoxifylline was applied with the nucleus pulposus (NP+PTX group). In control animals nucleus pulposus was applied with a piece of Spongel containing 20 microL of physiologic saline solution in a similar fashion (NP+PS group). Endoneurial fluid pressure was recorded with a servo-null micropipette system using glass micropipettes with tip diameters of 4 microm. Endoneurial fluid pressure in the dorsal root ganglion was measured before and 3 hours after application of test substances. After measurement of endoneurial fluid pressure, the nerve root and dorsal root ganglion were processed for histology and evaluated by light microscope. Values of endoneurial fluid pressure before application of test substances were as follows: 2.4 +/- 1.2 cm H2O in the NP+PS (control) group and 1.8 +/- 0.4 cm H2O in the NP+PTX group. There was no statistically significant difference between these two pretreatment measurements. However, values of endoneurial fluid pressure after application were as follows: 8.6 +/- 1.8 cm H2O in the NP+PS group and 2.9 +/- 0.8 cm H2O in the NP+PTX group. Values of endoneurial fluid pressure in the NP+PTX group were significantly lower compared with the NP+PS group. Histologic examination consistently showed only a slight degree of edema evident in the NP+PTX group compared with the NP+PS group. Pentoxifylline, an anti-tumor necrosis factor-alpha drug, prevented the dorsal root ganglion compartment syndrome caused by topical application of nucleus pulposus. Anti-inflammatory cytokine therapy may become an effective treatment of sciatica due to disc herniation.

Neurochemical relationships in the action of opioid analgesics on cerebral cortex.

The experiments with unrestrained cats showed that opioid analgesics in near-analgesic doses decreased the amplitude of the primary test response recorded in the second sensorimotor zone of the cerebral cortex in 20-150-msec interval between the stimuli applied to thalamocortical fibers radiating from thalamic n. VPL. Application of test substances affecting certain neurotransmitter processes and microionophoretic application of drugs and neurotransmitters to cortical neurons showed that the inhibitory effect of opioid analgesics on cerebral cortex is probably realized through GABA-, serotonin-, beta-adreno-, and cholinergic structures. The excitatory amino acids are also involved into this process.

A Continuous Flow System (CFS) for chronic exposure of aquatic organisms

Effect studies on marine life are changing focus from acute lethal effects to long term sublethal effects. There is a need for laboratory test systems that can facilitate continuous supplies of test substances at sublethal concentrations. Stability and reproducibility of the test solutions and application of test substances in mixtures are required. With the Continuous Flow System (CFS) described, the aim has been to make a flexible tool adjustable to a wide range of studies. Thus, test solutions and dispersions of substances poorly miscible in water can be made in various ways, using different methods and devices for substance injection, mixing and further conditioning. More than one species of organisms can be tested simultaneously. The system can be run both with freshwater and seawater. Presently the system is used for bioaccumulation and biomarker studies related to impacts of oil industry discharges of produced water off shore. This paper presents the main features and functional parts of the system and some result examples.

Development of an in Vitro Method for Skin Corrosion Testing

National and international regulations require that chemicals must be properly classified, labeled, packaged, and transported based on their ability to damage or destroy tissue, e.g., skin. Traditionally, skin corrosion assessments were based on tests involving topical application of test substances to the skin of rabbits. In the present work, an in vitro skin corrosion test based on the use of reconstructed human skin cultures was developed as a potential replacement for in vivo rabbit skin tests for corrosion. In the in vitro method, test substances were applied topically to the stratum corneum surface of human skin cultures. Skin culture damage or cytotoxicity was measured as decreased 3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyltetrazolium bromide (MTT) vital dye metabolism. In time-course experiments, the time (in minutes) of test material exposure eliciting a 50% reduction of MTT metabolism (i.e., t50 value) was calculated. Using this method we evaluated 24 chemicals and found that the 9 corrosive chemicals were accurately distinguished from 15 strong, moderate, or mild skin irritants, using an in vitro cutoff of t50 < 3 min. Histologic examination of the cultures indicated gradations of epidermal necrosis quantitated using a specially designed grading scale, which correlated well with the corrosivity of treatment chemicals and cytotoxicity measurements. The predictivity of the method was confirmed and was consistent in skin culture models from two suppliers. Thus, the utility of human skin equivalent cultures as a screening tool for prediction of skin corrosivity appears to be independent of the commercial source of the cultures. We conclude that the in vitro assay using human skin equivalent cultures is a promising alternative to in vivo rabbit skin corrosion tests for assessment of the corrosivity of chemicals to human skin.

Laboratory handling and its influence on hormonal and metabolic parameters during acute inflammation in rats. A critique of long-term treatment by repeated injections.

Twice a day, rats were exposed to handling stress by sham i.p. injections during a period of five days. This procedure progressively drove up the basic blood levels of free adrenaline, noradrenaline and dopamine, while insulin decreased to below normal. Blood glucose returned successively from hyperglycemia during the beginning of the experiment to normal later on. If rats subjected to acute inflammation were handled, the parameters blood catecholamines, glucose and the lymphocyte ingress into the site of inflammation all showed the same patterns of changes, suggesting a certain synchronization of the metabolic events. This synchronization failed to occur in animals with inflammation, but without handling. In any case, the disturbed metabolism of handled animals did not normalize during the test period of five days. Therefore, application of test substances by repeated injections is a useless method for the correct investigation of chronic exogenous influences.

Cell detachment and growth of fibroblasts as parameters for cytotoxicity of inorganic metal salts in vitro.

Eight inorganic metal compounds (AlCl3, Al(OH)3 gel, Al(OH)3 salt, SnCl2, ZnSO4, K2Cr2O7, CdCl2, HgCl2) were tested for their cytotoxic effect on an established hamster fibroblast line (BHK-21/C13) in vitro using a cell detachment assay and two different growth assays, the cloning efficiency and the cell number after 2 days subconfluent culture as parameters. The test conditions for these assays were optimized, including incubation period, application of test substance, growth conditions and data analysis. Aluminum, zinc and tin compounds showed low cytotoxic effects when compared to potassium, cadmium and mercuric compounds. Potassium dichromate was highly toxic in both growth assays (0.0001-0.01 mM, with a clear dependency on the incubation time), whereas it proved to be only slightly toxic in the detachment assay (0.1-5 mM). Cadmium and mercuric chlorides were the most toxic compounds in the growth (0.00001-0.001 mM) and the cell detachment assays (0.01-0.1 mM). Variable incubation periods barely affected the cytotoxicity of mercuric chloride. Ranking of these cytotoxicity data was found to be identical to the ranking of LD50 values (oral, rat) as well as to the ranking according to threshold limit values for human workroom environment, and of human eye irritation data.