Tri 2-ethylhexyl Trimellitate Research Articles

In vitro and in silico approach to study the hormonal activities of the alternative plasticizer tri-(2-ethylhexyl) trimellitate TEHTM and its metabolites.

Tri-(2-ethylhexyl) trimellitate (TEHTM) is a plasticizer for polyvinyl chloride (PVC) material used in medical devices. It is an alternative to di-(2-ethylhexyl) phthalate (DEHP), a well-known reprotoxic and endocrine disruptor. As plasticizers are known to easily migrate when in contact with fatty biological fluids, patient exposure to TEHTM is highly probable. However, there is currently no data on the potential endocrine-disrupting effects of its human metabolites. To evaluate the effects of TEHTM metabolites on endocrine activity, they were first synthesized and their effects on estrogen, androgen and thyroid receptors, as well as steroid synthesis, were investigated by combining in vitro and in silico approaches. Among the primary metabolites, only 4-MEHTM (4-mono-(2-ethylhexyl) trimellitate) showed agonist activities on ERs and TRs, while three diesters were TR antagonists at non-cytotoxic concentrations. These results were completed by docking experiments which specified the ER and TR isoforms involved. A mixture of 2/1-MEHTM significantly increased the estradiol level and reduced the testosterone level in H295R cell culture supernatants. The oxidized secondary metabolites of TEHTM had no effect on ER, AR, TR receptors or on steroid hormone synthesis. Among the fourteen metabolites, these data showed that two of them (4-MEHTM and 2/1-MEHTM) induced effect on hormonal activities in vitro. However, by comparing the concentrations of the primary metabolites found in human urine with the active concentrations determined in bioassays, it can be suggested that the metabolites will not be active with regard to estrogen, androgen, thyroid receptors and steroidogenesis-mediated effects.

Plasticizer exposure of infants during cardiac surgery

In the present study we investigated the internal exposure situation of infant patients to the plasticizers TEHTM (tri-2-ethylhexyl trimellitate) and DEHP (di-2-ethylhexyl phthalate). The study collective included 21 infant patients aged 2–22 months that had to undergo cardiac surgery using cardio pulmonary bypass (CPB). Each patient, but one, received blood products during surgery. A special feature was that the used CPB tubings were exclusively plasticized with the alternative plasticizer TEHTM and were free of the standard plasticizer DEHP, that raises increasing toxicological concern. The blood products were stored in DEHP plasticized blood bags. Blood and urine samples of each infant patient were analysed before and after the surgery for the levels of the plasticizers DEHP and TEHTM and their metabolites. In general, the plasticizers were detected in the post-surgery blood samples only, with TEHTM in low levels (median 18.4 μg/L) and DEHP in rather elevated levels (median 1046 μg/L). With respect to the urine samples, TEHTM metabolites were not detected in any of the samples. DEHP metabolites were found in all urine samples, however, in significantly increased median levels in the post-surgery urine samples of the infants (increase factor 5–26). Thus, the present study clearly demonstrates the strong contribution of standard medical procedures to the internal plasticizer burden of patients. Particularly with regard to the suspected endocrine disrupting activities of the phthalate plasticizer DEHP, the elevated internal levels of this plasticizer and its metabolites in infants following cardiac surgery are alarming.

Human metabolism and kinetics of tri-(2-ethylhexyl) trimellitate (TEHTM) after oral administration.

Tri-(2-ethylhexyl) trimellitate (TEHTM) is a plasticizer for PVC material and is used for medical devices as an alternative to di-(2-ethylhexyl) phthalate. As plasticizers are known to migrate easily into contact liquids, exposure of patients to TEHTM is highly probable. In the present study, human metabolism pathways of TEHTM and its elimination kinetics were investigated. For that purpose, four healthy volunteers were orally exposed to a single dose of TEHTM. TEHTM and its postulated primary metabolites were investigated in blood samples (up to 48h after exposure), and in urine samples (collected until 72h after exposure) using liquid chromatography tandem mass spectrometry (LC-MS/MS). TEHTM was found to be regioselectively hydrolyzed to its diesters di-2-(ethylhexyl) trimellitates (1,2-DEHTM, 2,4-DEHTM) with maximum blood concentrations at 3-h post-exposure, and to its monoester isomers mono-2-(ethylhexyl) trimellitates (1-MEHTM, 2-MEHTM) with peak blood concentrations 5-h post-exposure. For the elimination of investigated urinary metabolites, biphasic elimination kinetics was observed. The most dominant urinary biomarker was found to be 2-MEHTM (2-mono-(2-ethylhexyl) trimellitate), followed by several specific secondary metabolites. All in all, approximately 5.8% of the orally administered dose was recovered in urine over a period of 72h, indicating a comparatively low resorption rate of TEHTM in humans in combination with an apparently rather slow metabolism and excretion rate. In fact, TEHTM and selected metabolites were still detectable in blood and urine 48-h and 72-h post-exposure, respectively. This study is the first to elucidate TEHTM metabolism pathways in humans and to identify metabolites of TEHTM in blood and urine by usage of especially designed human biomonitoring methods. Powerful tools for exposure monitoring and risk assessment of TEHTM are therewith available for future research.

Comprehensive monitoring of specific metabolites of tri-(2-ethylhexyl) trimellitate (TEHTM) in urine by column-switching liquid chromatography-tandem mass spectrometry.

Tri-(2-ethylhexyl) trimellitate (TOTM or TEHTM) is a substitute for the plasticizer di-(2-ethylhexyl) phthalate (DEHP). Here, a fast and robust HPLC method is presented for the first time enabling the simultaneous quantification of several TEHTM metabolites in urine. These are the three TEHTM monoester isomers 1-mono-(2-ethylhexyl) trimellitate (1-MEHTM), 2-mono-(2-ethylhexyl) trimellitate (2-MEHTM), and 4-mono-(2-ethylhexyl) trimellitate (4-MEHTM) as well as several selected side chain oxidized monoesters of TEHTM, namely, 1-mono-(2-ethyl-5-hydroxyhexyl) trimellitate (5OH-1-MEHTM), 2-mono-(2-ethyl-5-hydroxyhexyl) trimellitate (5OH-2-MEHTM), 1-mono-(2-ethyl-5-oxohexyl) trimellitate (5oxo-1-MEHTM), 2-mono-(2-ethyl-5-oxohexyl) trimellitate (5oxo-2-MEHTM), 1-mono-(2-ethyl-5-carboxypentyl) trimellitate (5cx-1-MEPTM), 2-mono-(2-ethyl-5-carboxypentyl) trimellitate (5cx-2-MEPTM), 2-mono-(2-carboxymethylhexyl) trimellitate (2cx-2-MMHTM), and 1-mono-(2-carboxymethylhexyl) trimellitate (2cx-1-MMHTM). The method is characterized by a short sample preparation, for which the urine samples are enzymatically hydrolyzed and cleaned up by an online column arrangement. Separation of the analytes is enabled using liquid chromatography coupled with tandem mass spectrometry. Thus, in less than 30min, 11 postulated metabolites of TEHTM can be selectively and sensitively quantified. The method is distinguished by its wide linear working range of up to 1800μg/L with detection limits ranging from 0.3μg/L (for 5oxo-1-MEHTM) to 1.5μg/L (for 1-MEHTM). Precision and repeatability of the method were proven with determined relative standard deviations between 2.5 and 11.3%. The selection of the analytes of this method was based on a pilot study, by which several potential TEHTM metabolites were investigated in human urine of an orally exposed volunteer. Thus, the here presented method is a perfect tool for human biomonitoring of TEHTM exposure. Graphical abstract Analysis of several postulated TEHTM metabolites in human urine using LC-MS/MS.

Regioselective ester cleavage of di-(2-ethylhexyl) trimellitates by porcine liver esterase

In a comparative study the ester hydrolysis of the plasticizers di-(2-ethylhexyl) phthalate (DEHP) and tri-(2-ethylhexyl) trimellitate (TEHTM) as well as of the diester isomers 1,2-di-(2-ethylhexyl) trimellitate (1,2-DEHTM), 1,4-di-(2-ethylhexyl) trimellitate (1,4-DEHTM) and 2,4-di-(2-ethylhexyl) trimellitate (2,4-DEHTM) was investigated by a newly developed in vitro experimental design using porcine liver esterase (PLE). The substrates were incubated with PLE for 48h at 25°C in borate buffer and samples were taken at predetermined intervals during the experiment. The samples were processed using liquid-liquid extraction and analyzed using LC-MS/MS. The results demonstrated a rapid and extensive hydrolysis of the diester DEHP to the monoester mono-(2-ethylhexyl) phthalate (MEHP) during the incubation with PLE. The isomers of DEHTM were also hydrolyzed by PLE to a high extent, whereas TEHTM showed a high stability against enzymatic hydrolysis. The regioselective analysis revealed that the monoester isomers 1-MEHTM and 2-MEHTM were predominantly produced during the degradation of DEHTM isomers, indicating a preferred hydrolysis at the para-position. These findings are eminent for planning further investigations on the human TEHTM metabolism, as the extent, rate and route of metabolism are of crucial importance for a toxicological assessment.

A study to support regulatory submission assessing effects of Tri(2-ethylhexyl) trimellitate (TEHTM) upon pre and post-natal development in the rat, with a comparative group receiving Di(2-ethyl hexyl) phthalate (DEHP) included

Oral exposure of rodents to the plasticizer, Di(2-ethyl hexyl) phthalate (DEHP), elicits birth defects and effects upon the reproductive tract. There is currently limited published information regarding potential developmental effects in rodents of another phthalate ester, TEHTM.

Molecular modelling of phthalates – PPARs interactions

Di(2-ethylhexyl) phthalate (DEHP) is the most widely plasticizer for polyvinyl chloride (PVC) that is used in plastic tubes, in medical and paramedical devices as well as in food storage packaging. The toxicological profile of DEHP has been evaluated in a number of experimental animal models and has been extensively documented. Its toxicity is in part linked to the activation of the peroxisome proliferator-activated receptor α (PPARα). As a response, an intensive research for a new, biologically inert plasticizer has been initiated. Among the alternative studied, tri(2-ethylhexyl) trimellitate (TEHTM) or trioctyl trimellitate (TOTM) has attracted increasing interest. However, very little information is available on their biological effects. We proceeded to dock TOTM, DEHP and its metabolites in order to identify compounds that are likely to interact with PPARα and PPARγ binding sites. The results obtained hint that TOTM is not able to bind to PPARs and should therefore be safer than DEHP.

Blood response to plasticized poly(vinyl chloride): dependence of fibrinogen adsorption on plasticizer selection and surface plasticizer level.

The high level of plasticizer in plasticized poly(vinyl chloride) (PVC) ensures that plasticizer selection has an important influence on the suitability of PVC to function in blood-contacting applications. In this study, three types of plasticized PVC in sheet form, with di-(2-ethylhexyl)phthalate (DEHP), tri-(2-ethylhexyl)trimellitate (TEHTM) and n-butyryltri-n-hexyl citrate (BTHC) as plasticizer, were selected for assessment and single solute fibrinogen adsorption was utilized as an initial index of interactions with blood components. Fibrinogen adsorption behavior shows a strong dependence on the plasticizer selection, plasticizer level at the surface and the adsorption conditions, such as adsorption time and fibrinogen solution concentration. Results indicate that BTHC plasticized PVC possesses the lowest adsorption capacity in the three types of plasticized PVC, while TEHTM plasticized PVC seems to have the strongest reactivity in certain fibrinogen solution concentrations. The alteration of surface plasticizer level was achieved by a methanol-cleaning treatment with a variety of cleaning times and the fibrinogen adsorption on plasticized PVC decreases with the reduction of surface plasticizer level. The migration behavior of two phthalate esters (DEHP and TEHTM) was evaluated using UV-Spectrophotometer to determine the plasticizer level at the surfaces. In addition, the fibrinogen adsorption mechanism was examined with Freundlich adsorption modeling.

Comparative study of the leachability of di(2-ethylhexyl) phthalate and tri(2-ethylhexyl) trimellitate from haemodialysis tubing

The leachability of both Di(2-ethylhexyl) phthalate (DEHP) and Tri(2-ethylhexyl) trimellitate (TEHTM) or Trioctyl trimellitate (TOTM) from haemodialysis tubing was investigated in 20 patients with chronic renal failure undergoing maintenance haemodialysis. The blood tubing made of common polyvinyl chloride (PVC) plasticized with DEHP (group 1 patients) were replaced with tubing plasticized with TOTM-DEHP (group 2 patients). The patient blood obtained from the inlet and the outlet of the dialyzer was analyzed during a 4 h-dialysis session. Thus, the circulating concentrations of both DEHP and TOTM resulting from the release from dialyzer tubes were estimated using High-performance Liquid chromatograph (HPLC). With the common PVC-DEHP blood tubing, a DEHP quantity of 122.95±33.94 mg was extracted from tubing during a single dialysis session (ranging from 55 to 166.21 mg). During the same period, the total amounts of DEHP retained by the patients were 27.30±9.22 mg (ranging from 12.50 to 42.72 mg). As for blood tubing plasticized with TOTM-DEHP, 41.80±4.47 mg of DEHP and 75.11±25.72 mg of TOTM were extracted. During the same period, the amounts of DEHP and TOTM retained by the patients were 3.42±1.37 mg and 4.87±2.60 mg, respectively. The extraction rate both plasticizers was correlated with serum lipid content (cholesterol+triglyceride) ( r 2=0.75 for DEHP and r 2=0.64 for TOTM). In the present investigation, less TOTM and DEHP were apparently released from haemodialysis tubing plasticized with TOTM-DEHP than DEHP released from haemodialysis tubing plasticized with DEHP only. TOTM seems to be a superior alternative to DEHP for use in medical devices because of its potential lower leachability. To recommend it as an alternative plasticizer, its possible toxicity towards human body should be investigated before it can be used routinely. However, patients undergoing haemodialysis using tubing plasticized with DEHP only are regularly exposed to non negligible amounts of DEHP. In view of several biological effects previously reported, it is time to reconsider the use of DEHP only as a plasticizer.

Correlation of cytokine elaboration with mononuclear cell adhesion to platelet storage bag plastic polymers: a pilot study.

The basis for many febrile nonhemolytic transfusion reactions associated with platelet transfusion therapy is cytokine elaboration and accumulation in the storage bag, which correlate with the leukocyte content and the length of platelet storage. We propose that a possible additional variable in the elaboration and accumulation of cytokines is the differential adhesion of mononuclear cells to the plastic substrate of the platelet storage bag. We hypothesize that mononuclear cell adhesion-induced cytokine release is greater in random-donor platelet bags composed of the polyolefin polymer compared to the single-donor apheresis platelet bags composed of the polyvinyl chloride polymer with the tri-(2-ethylhexyl) trimellitate (TEHTM) plasticizer. For four blood donors, we demonstrate preferential mononuclear cell adhesion, in vitro, to discs of polyolefin polymer versus discs of polyvinyl chloride polymer with the TEHTM plasticizer. Scanning electron microscopy corroborates this. In addition, proinflammatory cytokine (interleukin 1beta [IL-1beta] and tumor necrosis factor alpha [TNF-alpha]) levels are greater in culture wells containing discs of polyolefin polymer than in those containing discs of polyvinyl chloride polymer with the TEHTM plasticizer, and even more so in storage bags containing polyolefin polymer versus polyvinyl chloride polymer with the TEHTM plasticizer (IL-1beta, TNF-alpha, IL-6, and IL-8). This study suggests, for the first time, that differential plastic substrate mononuclear cell adhesion may contribute to cytokine release during platelet storage. This may represent an additional variable in the pathophysiology of febrile nonhemolytic transfusion reactions in patients receiving stored platelet units.

Blood interactions with plasticized poly(vinyl chloride): relevance of plasticizer selection.

An investigation has been made of blood interactions with plasticized poly(vinyl chloride) (PVC) biomaterials in tubular form, taking into account the influence on the blood response of the polymer, antithrombotic agent, blood condition and test procedure. In vitro and ex vivo procedures were used to achieve a comparison between PVC plasticized with di- (2-ethylhexyl)phthalate (DEHP) and with tri-(2-ethylhexyl)trimellitate (TEHTM). The blood response was monitored in terms of the measurement of fibrinogen adsorption capacity, thrombin-antithrombin III complex (TAT) and the complement component C3a. Surface characterization of the polymers was performed by X-ray photoelectron spectroscopy (XPS). The data obtained indicate that in comparison with DEHP-PVC, there is a higher reactivity for TEHTM-PVC, which correlates with the plasticizer distribution at the polymer surface.

Characteristics of white cell-reduced red cells stored in tri-(2-ethylhexyl)trimellitate plastic.

Standard blood storage containers contain extractable plasticizers that accumulate in blood during storage and are an unintended transfusion product. However, extractable plasticizers have a protective effect on the red cell membrane and improve red cell storage variables. Prestorage white cell reduction also improves selected red cell storage variables. The study evaluated whether the beneficial effect of prestorage white cell reduction would offset the negative effect of the absence of extractable plasticizer in red cells stored in AS-3 for 42 days at 4 degrees C. Filtered red cells stored in polyvinylchloride containers with the nonextracting plasticizer, tri-(2-ethylhexyl)trimellitate (TEHTM), were compared to unfiltered red cells stored in polyvinylchloride containers with the extractable plasticizer di-(2-ethylhexyl)phthalate (DEHP). Poststorage supernatant potassium and red cell osmotic fragility were significantly higher in white cell-reduced TEHTM units than in unfiltered DEHP units. The mean 24-hour recovery of the filtered TEHTM red cells was significantly lower than that of the unfiltered DEHP red cells (69.1 +/- 7.4% vs. 77.1 +/- 5.1%, p < 0.05, n = 8). These data demonstrate that white cell reduction before 42-day storage in TEHTM containers with currently approved preservatives does not yield an acceptable red cell component.

Tissue distribution and excretion of tri-(2-ethylhexyl)trimellitate in rats.

The disposition kinetics of tri-(2-ethylhexyl)trimellitate (TEHTM), a new plasticizer for polyvinyl chloride (PVC) plastic, was studied in rats following intravenous administration of [14C-carbonyl]tri-(2-ethylhexyl)trimellitate using an oil in water emulsion as the vehicle. The distribution half-life, elimination half-life, and clearance values estimated from the plasma concentration of radioactivity data obtained following iv administration of 10.5 mg/kg of TEHTM (59.9 muCi/kg), were 46.2 min, 5.34 d, and 40.5 ml/kg X h, respectively. Following iv dosage of 15.6 mg/kg of TEHTM (28.0 muCi/kg), significant accumulation of radioactivity was found in the liver, lungs, and spleen, with liver accounting for 72% of the administered dosage at 24 h. Excretion of TEHTM and its biotransformation products was slow, with 21.3% of the administered radioactivity found in the feces and 2.8% in the urine during the 14-d collection period. Biliary excretion seems to be the major route of elimination of TEHTM. The pharmacokinetic data gathered in the present investigation are compared to di-(2-ethylhexyl)phthalate (DEHP), a widely used plasticizer for PVC.

Contamination of platelet storage bags by phthalate esters.

Phthalate esters are the most extensively used plasticizers in the manufacture of polyvinylchloride (PVC) plastic. Many medical devices used in the collection and storage of blood components are made of PVC plastic containing di(2-ethylhexyl) phthalate (DEHP). DEHP leaches at a rate of 100 micrograms/ml X d into platelet concentrate (PC) supernatant when PCs are stored in PVC containers. It is only possible to store PCs for 72 h in this DEHP plastic, after which time the platelet function has deteriorated and they cannot be used for transfusion therapy. Since it was desirable to find a container that permitted longer storage times and because of the concern for the toxicity of DEHP, new bags, manufactured with different plastic formulations without this plasticizer, were tested for PC storage. Using these new containers, such as the PL732 [polyolefin (PO) plastic], and the CLX300 and PL1240 [tri(2-ethylhexyl) trimellitate (TEHTM) PVC plastic], it was possible to store PCs for 5 d while preserving platelet function. In spite of these new plastic bags being manufactured without DEHP, we found DEHP and its metabolite mono(2-ethylhexyl) phthalate (MEHP) as contaminants of the supernatant of the PCs stored in these containers. After analyzing the plastic material of each of these containers, we were able to identify the source of the contamination as coming from the plastic materials that were used in the manufacture of the bags. The sterilization process of the PL732 bag was investigated, since it was found that when the plastic of the PL732 bag was analyzed prior to sterilization, no contamination by DEHP was detected; however, whether the PL732 bag was sterilized together with the primary PVC bag or separately, using ethylene oxide, contamination by DEHP was found, suggesting contamination of the sterilization unit by DEHP.

Bacterial mutagenicity testing of urine from rats dosed with 2-ethylhexanol derived plasticizers

Di-(2-ethylhexyl)phthalate (DEHP) produced hepatocellular carcinomas in rodents at high doses in a NTP/NCI bioassay. DEHP has not shown evidence of genotoxic activity in in vitro mutagenecity tests. We extended these studies by examining the mutagenicity of urine from rats dosed with DEHP, 2-ethylhexanol (2-EH), and several other 2-EH derived plasticizers, i.e. di-(2-ethylhexyl)adipate (DEHA), di-(2-ethylhexyl)terephthalate (DEHT) and tri-(2-ethylhexyl)trimellitate (TEHT). A modified Ames Salmonella/microsome assay was used to determine mutagenicity. Urine was pooled from male Sprague-Dawley rats dosed daily for 15 days with 2000 mg/kg of each test substance with the exception of 2-EH which was given at 1000 mg/kg. Direct plating procedures were used to determine the presence of mutagens in urine. Urine from rats dosed with 8-hydroxyquinoline was used as a positive control. There was no evidence that mutagenic substances were excreted in the urine by rats dosed with either DEHP, DEHA, DEHT, TEHT or 2-EH as determined in the presence or absence of rat liver microsomes, and with or without treatment with β-glucuronidase/aryl sulfatase. Our findings indicate that the above test compounds were not converted to urinary metabolites that were mutagenic. These observations provide no evidence for a genotoxic mechanism for DEHP carcinogenicity in rodents.