Qualitative and Quantitative Analysis of Certain Aspects of the Cytotoxic and Genotoxic Hazard of Hospital Wastewaters by Using a Range of In Vitro Assays.

Health care facilities and hospitals generate significant amounts of wastewater which are released into the sewage system, either after a preliminary treatment or without any further treatment. Hospital wastewater may contain large amounts of hazardous chemicals and pharmaceuticals, some of which cannot be eliminated entirely by wastewater treatment plants. Moreover, hospital effluents may be loaded with a plethora of pathogenic microorganisms or other microbiota and microbiome residues. The need to monitor hospital effluents for their genotoxic hazard is of high importance, as detailed information is scarce. DNA-based information can be acquired directly from samples through the application of various molecular methods, while cell-based biomonitoring assays can provide important information about impaired cellular pathways or mechanisms of toxicity without prior knowledge of the identity of each toxicant. In our study, we evaluated samples of chlorinated hospital wastewater discharged into the sewage system after this disinfection process. The assessment of cytotoxicity, genotoxicity and mutagenicity of the hospital effluents was performed in vitro by using a broad battery of biomonitoring assays that are relevant for human health effects. All the tested hospital wastewater samples could be classified as potentially genotoxic, and it is concluded that the microbiota present in hospital wastewater might contribute to this genotoxic potential.

Toxicological testing of a photoactive phthalocyanine-based antimicrobial substance.

The aim of the study was toxicological testing of an innovative and efficient antimicrobial agent based on photoactive phthalocyanine (Pc) derivative. A promising Aluminium phthalocyanine (AlPc) with efficient and stable antimicrobial effects was subjected to a battery of toxicological tests to avoid local and systemic toxicity hazard. In compliance with the current European legislation restricting the use of experimental animals, the methods comprised exclusively in vitro procedures based on cellular and tissue models of human origin or mimicking human tissues. The battery of toxicological tests to identify local toxicity included skin corrosion/irritation, eye irritation, and phototoxicity. The basic systemic toxicity tests included acute toxicity, skin sensitization, genotoxicity, and endocrine disruption. The results showed that AlPc induced skin and eye irritation, exhibited borderline sensitization potential and mutagenic potential in one test strain of the Ames test, which was not confirmed in the chromosome aberration test. The AlPc was found to be phototoxic. The results from the cytotoxicity test designed for acute oral toxicity estimation were not conclusive, the acute toxicity potential has to be determined by conventional tests in vivo. Regarding endocrine disruption, no agonistic activity of the AlPc on human estrogen receptor α, nor human androgen receptor was observed. The skin penetration/absorption test revealed that the AlPc has not penetrated into the dermis and receptor fluid, confirming no risk of systemic exposure via the bloodstream.

Toxicity of wastewater from health care facilities assessed by different bioassays.

OBJECTIVES: The purpose of this study was to determine toxicity of wastewater from hospitals in the Czech Republic using traditional and alternative toxicological methods. The pilot study comprised weekly dynamics of sewage ecotoxicity of treated wastewater from one hospital in two different seasons. A detailed investigation of wastewater ecotoxicity, genotoxicity and reprotoxicity followed in five different hospitals. METHODS: The seven following bioassays were used in this study: algal growth inhibition test (ISO 8692), Vibrio fischeri test (ISO 11348-2), Daphnia magna acute toxicity test (ISO 6341), Allium cepa assay, Ames test (OECD TG 471), Comet assay and YES/YAS assay. RESULTS: The wastewater ecotoxicity during one week showed no differences in separate working days, however, higher toxicity values were recorded in May compared to November. In the following study, samples from two of the five hospitals were classified as toxic, the others as non toxic. Genotoxicity has not been confirmed in any sample. In several cases, wastewater samples exhibited agonist activity to the estrogen and androgen receptors. CONCLUSION: The study demonstrated different levels of toxicity of treated hospital wastewater. Variable sensitivity of individual bioassays for tested wastewater samples was recognized. A more extensive study including proposal for improvement of hospital wastewater treatment within the Czech Republic can be recommended with the aim to decrease the discharge of toxic chemicals into the local sewage system and the environment.

Bioindicators of wastewater ecotoxicity.

Wastewater, especially containing hospital effluents, exhibits high chemical complexity and specificity since it includes various chemicals, biocides, pharmaceuticals, surfactants, radionuclides, disinfectants and pathogens. Biological tests provide true evidence of the wastewater quality and unlike chemical analytical tests show comprehensive pollution effects on the environment and human health. Normalized conventional bioassays are not sensitive enough for ecotoxicological evaluation of wastewater and there is a great need for the development of suitable sensitive bioassays in order to characterize properly the residual toxicity of treated effluents. Provisions of binding EU legislation regarding protection of animals used for scientific purposes and legislation dealing with test methods for identification and classification of health hazard of chemicals, pharmaceuticals, biocides, medical devices and consumer products such as cosmetics for environmental ecosystems and for man require to employ alternative toxicological methods respecting the 3Rs concept with priority given to methods in vitro. The Fish Embryo Test (FET) is identified as a relevant, reliable and efficient alternative test method in vitro for determination of acute toxicity for fish. Using the FET, additional toxicological endpoints may be investigated to assess organ specific bioaccumulation, genotoxicity and mutagenicity, developmental toxicity, teratogenicity, various forms of neurotoxicity or endocrine disruptivity. The addition of multiparametric sensitive endpoints makes the FET a true alternative in vitro assay and a powerful tool in toxicology.

Genotoxicity of wastewater from health care facilities.

Health care facilities use for therapeutic purposes, diagnostics, research, and disinfection a high number of chemical compounds, such as pharmaceuticals (e.g. antibiotics, cytostatics, antidepressants), disinfectants, surfactants, metals, radioactive elements, bleach preparations, etc. Hospitals consume significant amounts of water (in the range of 400 to 1200 liters/day/bed) corresponding to the amount of wastewater discharge. Some of these chemicals are not eliminated in wastewater treatment plants and are the source of pollution for surface and groundwater supplies. Hospital wastewater represents chemical and biological risks for public and environmental health as many of these compounds might be genotoxic and are suspected to contribute to the increased incidence of cancer observed during the last decades. The changes of the genetic information can have a lethal effect, but more often cause tumor processes or mutations in embryonic development causing serious defects. A review of the available literature on the mutagenicity/genotoxicity of medical facilities wastewater is presented in this article.

Low-cost and prototype-friendly method for biocompatible encapsulation of implantable electronics with epoxy overmolding, hermetic feedthroughs and P3HT coating.

The research of novel implantable medical devices is one of the most attractive, yet complex areas in the biomedical field. The design and development of sufficiently small devices working in an in vivo environment is challenging but successful encapsulation of such devices is even more so. Industry-standard methods using glass and titanium are too expensive and tedious, and epoxy or silicone encapsulation is prone to water ingress with cable feedthroughs being the most frequent point of failure. This paper describes a universal and straightforward method for reliable encapsulation of circuit boards that achieves ISO10993 compliance. A two-part PVDF mold was machined using a conventional 3-axis machining center. Then, the circuit board with a hermetic feedthrough was placed in the mold and epoxy resin was injected into the mold under pressure to fill the cavity. Finally, the biocompatibility was further enhanced with an inert P3HT polymer coating which can be easily formulated into an ink. The biocompatibility of the encapsulants was assessed according to ISO10993. The endurance of the presented solution compared to silicone potting and epoxy potting was assessed by submersion in phosphate-buffered saline solution at 37 °C. The proposed method showed superior results to PDMS and simple epoxy potting.

Sensitization potential of medical devices detected by in vitro and in vivo methods.

Medical devices must be tested before marketing in accordance with ISO EN 10993-10 in order to avoid skin sensi­tization. This standard predominantly refers to the in vivo test but does not exclude the use of in vitro methods that have been sufficiently technically and scientifically validated for medical device testing. It is foreseen that, due to the complexity of the sensitization endpoint, a combination of several methods will be needed to address all key events occurring in the sensitization process. The objective of this pilot study was to evaluate the sensitization potential of selected medical devices using a combination of in chemico (DPRA, OECD TG 442C) and in vitro (LuSens, OECD TG 442D) methods in comparison with the in vivo (LLNA DA, OECD TG 442A) method and to suggest a possible testing strategy for the safety assessment of medical device extracts. Overall, one of the 42 tested samples exhibited positive results in all employed test methods, while 33 samples were predicted as non-sensitizing in all three performed methods. This study demonstrated good agreement between in vitro and in vivo results regarding non-sensitizing samples; however, some discrepancies in positive classification were recorded. A testing strategy is suggested in which negative results are accepted and any positive results in the in chemico or in vitro tests are followed up with a third in vitro test and evaluated in accordance with the "2 out of 3 approach". This strategy may reduce and replace animal use for testing the sensitization potential of medical devices.

Novel lipophosphonoxin-loaded polycaprolactone electrospun nanofiber dressing reduces Staphylococcus aureus induced wound infection in mice.

Active wound dressings are attracting extensive attention in soft tissue repair and regeneration, including bacteria-infected skin wound healing. As the wide use of antibiotics leads to drug resistance we present here a new concept of wound dressings based on the polycaprolactone nanofiber scaffold (NANO) releasing second generation lipophosphonoxin (LPPO) as antibacterial agent. Firstly, we demonstrated in vitro that LPPO released from NANO exerted antibacterial activity while not impairing proliferation/differentiation of fibroblasts and keratinocytes. Secondly, using a mouse model we showed that NANO loaded with LPPO significantly reduced the Staphylococcus aureus counts in infected wounds as evaluated 7 days post-surgery. Furthermore, the rate of degradation and subsequent LPPO release in infected wounds was also facilitated by lytic enzymes secreted by inoculated bacteria. Finally, LPPO displayed negligible to no systemic absorption. In conclusion, the composite antibacterial NANO-LPPO-based dressing reduces the bacterial load and promotes skin repair, with the potential to treat wounds in clinical settings.

Extrapolation of toxic indices among test objects.

Oligochaeta Tubifex tubifex, fish fathead minnow (Pimephales promelas), hepatocytes isolated from rat liver and ciliated protozoan are absolutely different organisms and yet their acute toxicity indices correlate. Correlation equations for special effects were developed for a large heterogeneous series of compounds (QSAR, quantitative structure-activity relationships). Knowing those correlation equations and their statistic evaluation, one can extrapolate the toxic indices. The reason is that a common physicochemical property governs the biological effect, namely the partition coefficient between two unmissible phases, simulated generally by n-octanol and water. This may mean that the transport of chemicals towards a target is responsible for the magnitude of the effect, rather than reactivity, as one would assume suppose.