In vitro toxicity of infusion sets depends on their composition, storage time and storage conditions
Graphical abstract
Leachables from the rubber-made parts (injection site and flashball) of infusion sets were found to induce the highest in vitro toxicity, but leachables from other parts (tube and drip chamber) can also contribute to the overall toxic effects induced by infusion sets.
Introduction
Disposable medical devices (DMDs) such as infusion sets, catheters, blood bags, etc., are widely used in the clinical practice. While being considered safe by most healthcare providers, DMDs can release toxic compounds (toxic leachables) during their clinical use (Jenke et al., 2006, Paskiet et al., 2013, Schumacher et al., 2007). Specifically, natural rubber latex, that is a component of many infusion sets and other DMDs, may induce profound toxicity, including local inflammation, allergic reactions, systemic toxicity, and increased risk of infections (Cormio et al., 1993, Dakwar et al., 2012, Ikarashi et al., 1992, Pariente et al., 2000, Talja et al., 1985). Phthalate plasticizers (such as di(2-ethylhexyl) phthalate, DEHP; di-(ethylhexyl)-terephthalate; DEHT; di-iso-nonyl-1,2-cyclohexane-dicarboxylate, DINCH; trioctyl trimellitate, TOTM) can leach out from the polyvinyl chloride (PVC) parts of the DMDs, and induce local and systemic toxicity in a dose-dependent fashion (Feigal, 2002, Bernard et al., 2015).
Indeed, analytical investigation of the infusion set leachates identified presence of DEHP, along with other potentially toxic leachables (e.g., the butylated hydroxytoluene) and particles (Schumacher et al., 2007). In our previous study, we confirmed that the individual parts of the infusion sets release toxic leachables and reported a new method for in vitro safety assessment of the infusion set leachates (Dakwar et al., 2012). Subsequently, we investigated the mechanisms of toxicity of the infusion sets leachates in in vitro experimental settings, and revealed that the latex-made parts of the infusion sets induce cell death via oncosis (Kozlovskaya and Stepensky, 2015). Leachates from the other parts of the infusion sets were less toxic, and induced some biochemical changes that were usually not accompanied by cytotoxicity.
Our previous studies focused on the toxicity analysis of the individual samples of infusion sets. However, there are profound differences in the design and composition of the infusion sets produced by different manufacturers. Generally, infusion sets are composed of the drip chamber (that is connected to the fluid bag), tube, and may include a port for administration of additional drugs (injection site or flashball, Fig. 1). The two major vendors of infusion sets that are used in Israel differ in their design. One of the most commonly used designs of the infusion sets produced by Manufacturer A contains a small-size injection site (apparently made of the synthetic rubber; Fig. 1A). The design of the infusion sets produced by Manufacturer B changed over time, starting with the big latex-made flashball, that has been replaced with the synthetic rubber, or excluded to generate a model without a flashball (Fig. 1B–D). Obviously, these differences in the design of the infusion sets and in the composition of their individual parts can affect their safety and the risk of the toxicity due to the release of the leachable compounds.
The primary objective of this study was to reveal the relative in vitro toxicity of infusion sets from the leading vendors that are used in Israel (Fig. 1), and to determine whether this toxicity is affected by the design of the items and their storage time and storage conditions. To attain this objective, we sampled infusion sets of different designs from Manufacturers A and B and analyzed in vitro toxicity of the leachates prepared from these samples according to the guidelines specified by the regulatory authorities, such as The United States Pharmacopoeia (USP) (Anon., 2009b, Anon., 2009c) and International Organization for Standardization (ISO) (Anon., 2009a). According to these guidelines, leachates from the individual parts of the infusion sets are incubated with the L-929 mouse fibroblasts for 24 h, and toxic effects are defined as significant (by more than 30%) reduction in the cell metabolic activity (by the MTT test), or prominent changes in cell amount, proliferation, colony formation etc. (Anon., 2009a, Anon., 2009b).
However, the experimental settings of the in vitro toxicity tests that are recommended by the regulatory agencies are not optimized for the safety assessment of the infusion sets and may fail to reveal their relative toxicity (Dakwar et al., 2012). Specifically, the L-929 fibroblasts do not represent the cells that are exposed to the infusion sets leachables in the clinical settings (i.e., cells that are lining the blood vessels (endothelial cells) and those present in the bloodstream (cells of the lymphoid and myeloid origin)). In addition, the composition of the medium that is used for the preparation of the leachates can affect the content of the leaching compounds. Exposure of the cells to the leachates for the 24 h may fail to reveal the relative toxicity of the analyzed samples, and shorter or longer duration of exposure may be required. MTT test, that is recommended by some of the authorities as the experimental readout of the toxic effects, can lead to artefacts that confound assessment of the relative toxicity of the samples (e.g., signal higher than 100%) (Dakwar et al., 2012).
Therefore, our secondary objective in this study was to determine the most suitable experimental settings for analysis of the relative in vitro toxicity of infusion sets. In addition to the experiments with the L-929 cells, we applied the cEND and bEnd.3 brain capillary endothelial cells for toxicity testing of the studied samples. Moreover, we performed experiments with different composition of the cell culture medium (% of the fetal bovine serum, FBS), duration of cell incubation with the leachates (24 vs. 48 h), and the quantification of the toxic effects (microscopy, the MTT test, or neutral red assay).
Section snippets
Chemicals and materials
Cadmium chloride (CdCl2), hydrogen peroxide (H2O2), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), neutral red, phenol, propidium iodide (PI), sodium azide (NaN3), and tunicamycin (Tm) were obtained from Sigma–Aldrich Ltd. (Rehovot, Israel). Analytical grade DMSO was purchased from Bio-Lab Ltd. (Jerusalem, Israel).
List of the infusion sets samples that were analyzed in this study is presented in Table 1. The names of the manufacturers of the studied samples and their batch
Relative in vitro toxicity of the analyzed infusion sets
We determined the relative in vitro toxicity of the control solutions and of the individual parts of the studied infusion sets in the L-929, bEnd. 3 and cEND cells (Fig. 2). All the positive control solutions decreased significantly the metabolic activity of the cells (e.g., UV absorbance of 10.6 ± 2.7%, 7.9 ± 6.5% and 9.4 ± 2.3% for L-929, cEND and bEnd.3 cells, respectively, p < 0.01). All the 3 cell lines were sensitive to a similar extent to the toxic effects of the positive control solutions (the
Discussion
In our previous investigations, we identified deterioration in quality of infusion sets (change of color, release of potentially toxic chemicals) (Schumacher et al., 2007) and the relative toxicity of leachates from different parts of a single infusion sets batch (Dakwar et al., 2012). Moreover, we characterized sensitivity of selected cell lines (HeLa and cEND cells) to toxic leachates and revealed that cEND brain endothelial cell line can be used for in vitro toxicity analysis of DMDs
Conclusions
Infusion sets released toxic leachables and induce toxic effects on experimental cells during in vitro toxicity testing. Leachates from the rubber parts (flashball and injection site) were more toxic than those of the other parts of the infusion sets (tube and drip chamber). The extent of the measured toxicity was affected by the choice of the applied experimental settings: the cells, medium composition, exposure duration, and type of assay for toxicity assessment. We recommend to apply the
Acknowledgements
This work was supported by the research grant from the Israel Defense Forces Medical Corps and the Israeli Ministry of Defense (IMOD) Directorate of Defense Research and Development (DDR&D).
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