Airway inflammation and adjuvant effect after repeated airborne exposures to di-(2-ethylhexyl)phthalate and ovalbumin in BALB/c mice

BACKGROUND: Epidemiological studies have suggested an association between exposure to phthalate plasticizers, including di-(2-ethylhexyl)phthalate (DEHP), and increased prevalence of asthma, rhinitis or wheezing. Furthermore, studies in mice have demonstrated an adjuvant effect from DEHP after parenteral administration with the model allergen ovalbumin (OVA). OBJECTIVE: Exposures to DEHP were investigated for adjuvant effects and airway inflammation in a mouse inhalation model. METHODS: BALB/cJ mice were exposed to aerosols of 0.022-13 mg/m(3) DEHP and 0.14 mg/m(3) OVA 5 days/week for 2 weeks and thereafter weekly for 12 weeks. Mice exposed to OVA alone or OVA+Al(OH)(3) served as control groups. Finally, all groups were exposed to a nebulized 1% OVA solution on three consecutive days. Serum, bronchoalveolar lavage (BAL) fluid, and draining lymph nodes were collected 24h later. RESULTS: In the OVA+Al(OH)(3) group, significantly increased levels of OVA-specific IgE and IgG1 in serum as well as of eosinophils in BAL fluid were observed. DEHP affected OVA-specific IgG1 production in a concentration-dependent manner, whereas little effect was seen on IgE and IgG2a. Dose-dependent increases in inflammatory cells were observed in BAL fluids, leading to significantly higher lymphocyte, neutrophil and eosinophil numbers in the OVA+13 mg/m(3) DEHP group. Ex vivo cytokine secretion by cultures of draining lymph nodes suggested that DEHP has a mixed Th1/Th2 cytokine profile. CONCLUSION: Airborne DEHP is able to increase serum IgG1 and lung inflammatory cell levels, but only at very high concentrations. Realistic DEHP levels do not have an adjuvant effect or induce allergic lung inflammation in the present mouse model.     

The adjuvant effect of di-(2-ethylhexyl) phthalate is mediated through a PPARalpha-independent mechanism

Previous studies in BALB/c mice revealed an adjuvant effect of di-(2-ethylhexyl) phthalate (DEHP) to simultaneously administered ovalbumin. DEHP is the most commonly used phthalate plasticizer. In vivo formed metabolites of DEHP are peroxisome-proliferator-activated receptor (PPAR) ligands, a group of chemicals that may have immunomodulatory properties. To study whether the PPARalpha receptor was involved in the adjuvant effect of DEHP, PPARalpha-deficient 129/Sv mice were exposed intraperitoneally to a mixture of OVA and DEHP, and the OVA-specific IgE, IgG1 and IgG2a responses were compared to the corresponding responses in the wild-type strain. The study showed that the adjuvant mechanism of DEHP is mediated through a PPARalpha-independent mechanism. Compared to mice only given OVA, DEHP induced highly increased levels of OVA-specific IgG1 and IgG2a, both in the wild-type and in the PPARalpha knock-out strains, indicating that DEHP is a mixed Th1/Th2 adjuvant.     

Effects of mono-2-ethylhexyl phthalate on the respiratory tract in BALB/c mice

Airborne mono-2-ethylhexyl phthalate (MEHP) was studied for acute airway effects using a bioassay with BALB/c mice. Concentration- and time-dependent effects were obtained by continuous monitoring of the breathing pattern during exposure to 0.3-43.6 mg/m3 MEHP for 60 min. Additionally, inflammatory effects of MEHP were studied from bronchoalveolar lavage (BAL) fluid. MEHP showed no upper airway irritating effect. Lower airway irritation was apparent from a concentration-dependent decrease in tidal volume (shallow respiration) with a no-observed effect level (NOEL) of 0.3 mg/m3. The respiratory rate reached a maximum at about 8 mg/m3, demonstrating a rapid shallow breathing pattern. At concentrations above 4.9 mg/m3, the time of pause, another marker of lung irritation, increased concentration-dependently, resulting in a decrease in respiratory rate at high exposure levels. BAL fluid obtained from 0 to 72 hours after a 60 min exposure to 30 mg/m3 MEHP showed that the number of macrophage reached maximum about 16 hours after exposure. The NOEL was 1.7 mg/m3. BAL content of neutrophils, lymphocytes, eosinophils and epithelial cells was normal after exposure to 30 or 1.7 mg/m3 MEHP. Based on worst case inhalation scenario in the general population, no airway irritation is expected from non-occupational levels of MEHP originating from DEHP.     

Does lipophilicity per se induce adjuvant effects? Methyl palmitate as model substance does not affect ovalbumin sensitization

Anthopogenically introduced substances and pollutants are suspected to promote sensitization and development of allergic airway diseases, that is, acting as adjuvants. Lipophilicity may serve as an immunological warning signal, promoting adjuvant effects. Whether the lipophilicity of an inhaled compound induces immunomodulatory effects was investigated in a murine inhalation model with the highly lipophilic methyl palmitate (MP) as model substance. First, studies of acute effects following a 1-h exposure of up to 348 mg/m3 MP showed no effects on cell composition in bronchoalveolar lavage (BAL) or on lung function parameters. Thus, MP did not possess irritant or inflammatory properties, which may be a precursive stimulus for adjuvant effects. Second, mice were exposed to aerosols of MP, 6 or 323 mg/m3, for 1 h followed by a 20-min low-dose ovalbumin (OVA) inhalation. OVA only and OVA + Al(OH)3 served as control groups. Exposures were performed 5 times/wk for 2 wk followed by a weekly exposure for 10 wk. Finally, the mice were challenged with a high-dose OVA aerosol for 3 consecutive days. Neither OVA-specific immunoglobulin (Ig) G1, IgE, or IgG2a production, nor inflammatory cells in BAL, nor respiratory patterns were significantly affected in the MP groups. The OVA + Al(OH)3 group had a significantly higher IgG1 and IgE production, as well as higher eosinophil infiltration in the BAL fluid. These studies showed that effects of adjuvants not are necessarily due to their lipophilicity; that is, additional structural properties are required.     

Urinary and amniotic fluid levels of phthalate monoesters in rats after the oral administration of di(2-ethylhexyl) phthalate and di-n-butyl phthalate

Two studies were designed to examine amniotic fluid and maternal urine concentrations of the di(2-ethylhexyl) phthalate (DEHP) metabolite mono(2-ethylhexyl) phthalate (MEHP) and the di-n-butyl phthalate (DBP) metabolite monobutyl phthalate (MBP) after administration of DEHP and DBP during pregnancy. In the first study, pregnant Sprague-Dawley rats were administered 0, 11, 33, 100, or 300 mg DEHP/kg/day by oral gavage starting on gestational day (GD) 7. In the second study, DBP was administered by oral gavage to pregnant Sprague-Dawley rats at doses of 0, 100, or 250 mg/kg/day starting on GD 13. Maternal urine and amniotic fluid were collected and analyzed to determine the free and glucuronidated levels of MEHP and MBP. In urine, MEHP and MBP were mostly glucuronidated. By contrast, free MEHP and free MBP predominated in amniotic fluid. Statistically significant correlations were found between maternal DEHP dose and total maternal urinary MEHP (p=0.0117), and between maternal DEHP dose and total amniotic fluid MEHP levels (p=0.0021). Total maternal urinary MEHP and total amniotic fluid MEHP levels were correlated (Pearson correlation coefficient=0.968). Statistically significant differences were found in amniotic MBP levels between animals within the same DBP dose treatment group (p<0.0001) and between animals in different dose treatment groups (p<0.0001). Amniotic fluid MBP levels increased with increasing DBP doses, and high variability in maternal urinary levels of MBP between rats was observed. Although no firm conclusions could be drawn from the urinary MBP data, the MEHP results suggest that maternal urinary MEHP levels may be useful surrogate markers for fetal exposure to DEHP.     

Effects of maternal exposure to di(2-ethylhexyl)phthalate (DEHP) during pregnancy on susceptibility to neonatal asthma

Di(2-ethylhexyl) phthalate (DEHP) is used as a plasticizer and is widely dispersed in the environment. In this study, we investigated the effects of maternal exposure to DEHP during pregnancy on neonatal asthma susceptibility using a murine model of asthma induced by ovalbumin (OVA). Pregnant BALB/c mice received DEHP from gestation day 13 to lactation day 21. Their offspring were sensitized on postnatal days (PNDs) 9 and 15 by intraperitoneal injection of 0.5 μg OVA with 200 μg aluminum hydroxide. On PNDs 22, 23 and 24, live pups received an airway challenge of OVA for 30 min. Offspring from pregnant mice that received DEHP showed reductions in inflammatory cell count, interleukin (IL)-4, IL-13, and eotaxin in their bronchoalveolar lavage fluid and in total immunoglobulin E and OVA-specific IgE in their plasma compared with offspring from pregnant mice that did not receive DEHP treatment. These results were consistent with histological analysis and immunoblotting. Maternal exposure to DEHP reduces airway inflammation and mucus production in offspring, with a decrease in inducible nitric oxide synthase (iNOS) in the lung tissue. This study suggests that maternal exposure to DEHP during pregnancy reduces asthmatic responses induced by OVA challenge in offspring. These effects were considered to be closely related to the suppression of Th2 immune responses and iNOS expression.     

Adjuvant and immuno-suppressive effect of six monophthalates in a subcutaneous injection model with BALB/c mice.

The prevalence of allergic airway diseases is rapidly increasing in Western Europe and North America. This increase in disease prevalence may be associated with environmental pollutants. The present study investigated the adjuvant and immuno-suppressive effect of a series of monophthalates which are considered to be important metabolites of commonly used phthalate plasticizers. The effects were studied in a screening model. Ovalbumin (OA), used as the model antigen, was injected subcutaneously in the neck region of BALB/cJ mice with or without one of the test substances, mono-n-butyl phthalate (MnBP), monobenzyl phthalate (MBnP), mono-n-octyl phthalate (MnOP), mono-2-ethylhexyl phthalate (MEHP), mono-iso-nonyl phthalate (MiNP) or mono-iso-decyl phthalate (MiDP). The levels of OA-specific IgE, IgG1 and IgG2a in sera were measured by ELISA. Immuno-suppressive effect, defined as a statistically significant reduction in IgE or IgG1 antibody production, was observed with MEHP (1000 microg/ml, IgE and IgG1), MnOP (1000 microg/ml, IgE and IgG1), MiNP (1000 microg/ml, IgE and 10 microg/ml, IgG1) and MiDP (100 microg/ml, IgE and IgG1). Adjuvant effect, defined as a statistically significant increase in IgE or IgG1 antibody level, occurred with MEHP (10 microg/ml, IgE), MnOP (100 microg/ml, and 10 microg/ml, IgG1) and MiNP (100 microg/ml, IgE). No statistically significant immune modulating effect was seen with MBnP and MnBP.     

Effects of di-(2-ethylhexyl) phthalate and five of its metabolites on rat testis in vivo and in in vitro

In an attempt to establish which compound or compounds are responsible for the testicular damage observed after administration of di-(2-ethylhexyl) phthalate (DEHP) in rats, the effects of the parent compound and five of its major metabolites (mono-(2-ethylhexyl) phthalate (MEHP), 2-ethylhexanol (2-EH), mono-(5-carboxy-2-ethylpentyl) phthalate, mono-(2-ethyl-5-oxohexyl) phthalate and mono-(2-ethyl-5-hydroxyhexyl) phthalate) were investigated in vivo and in vitro. The concentrations of MEHP and the three MEHP-derived metabolites in plasma were determined after single and multiple oral doses of DEHP. The plasma concentrations and areas under the plasma concentration-time curves (AUC's) of each of the MEHP-derived metabolites were considerably lower than those of MEHP both after single and after repeated administration of 2.7 mmol of DEHP/kg body weight. The mean elimination half-life of MEHP was significantly shorter in animals given repetitive doses than in those given a single dose, but there was no statistically significant difference between the mean AUC values. No testicular damage was observed in young rats given oral doses of 2.7 mmol of DEHP or 2-EH/kg body weight daily for five days. In animals which received corresponding doses of MEHP the number of degenerated spermatocytes and spermatids was increased, whereas no such effects were found in animals given the MEHP-derived metabolites. MEHP was also the only compound that enhanced germ cell detachment from mixed primary cultures of Sertoli and germ cells.     

No background concentrations of di(2-ethylhexyl) phthalate and mono(2-ethylhexyl) phthalate in blood of rats

We measured the background levels of di(2-ethylhexyl) phthalate (DEHP) and its hydrolytic metabolite mono(2-ethylhexyl) phthalate (MEHP) in blood from naive female Sprague-Dawley rats and in de-ionized charcoal-purified water using an analytical procedure that is based on sample treatment with acetonitrile, n-hexane extraction and analysis by gas chromatography. In blood, blank values of 91.3 +/- 34.7 micrograms DEHP/l (n = 31) and 30.1 +/- 13.1 micrograms MEHP/l (n = 20) were obtained, and in water, values of 91.6 +/- 44.2 micrograms DEHP/l (n = 26) and 26.7 +/- 10.4 micrograms MEHP/l (n = 15) were found. Since there is no difference between the background valves obtained from blood of naive rats and water, we conclude that DEHP and MEHP result from contamination during the analytical procedure.     

Assessment of the teratogenicity of di(2-ethylhexyl)phthalate and mono(2-ethylhexyl)phthalate in mice

Di(2-ethylhexyl)phthalate (DEHP) and mono(2-ethylhexyl)phthalate (MEHP) were administered PO or IP to pregnant ICR mice at varying doses on days 7, 8, and 9 of gestation. In groups given DEHP orally, resorptions and malformed fetuses increased significantly at 1,000 mg/kg. Fetal weights were also significantly suppressed. Anterior neural tube defects (anencephaly and exencephaly) were the malformations most commonly produced. No teratogenic effects were revealed by IP doses of DEHP and PO or IP doses of MEHP, although high doses were abortifacient and lethal to pregnant females. Thus DEHP is highly embryotoxic and teratogenic in mice when given PO but not IP. The difference in metabolism, disposition, or excretion by the route of administration may be responsible for the difference in DEHP teratogenicity. Although MEHP is a principal metabolite of DEHP and is several times more toxic than DEHP to adult mice, it seems that MEHP and its metabolites are not teratogenic in ICR mice.     

Toxicokinetic relationship between di(2-ethylhexyl) phthalate (DEHP) and mono(2-ethylhexyl) phthalate in rats

The toxicokinetic relationship between di(2-ethylhexyl) phthalate (DEHP) and mono(2-ethylhexyl) phthalate (MEHP), a major metabolite of DEHP, was investigated in Sprague-Dawley rats orally treated with a single dose of 14C-DEHP. Urinary excretion of total 14C-DEHP and of its metabolites was followed by liquid scintillation counting (LSC). Concentrations of DEHP and MEHP were determined 6, 24, and 48 h after treatment in rat serum and 6, 12, 24, and 48 h after treatment in urine by high-performance liquid chromatography (HPLC). After 24 h, peak concentrations of MEHP in both urine and serum were observed in animals treated with 40, 200, or 1000 mg DEHP/kg. HPLC showed that general toxicokinetic parameters, such as Tmax (h), Cmax (microg/ml), Ke (1/h), and AUC (microg-h/ml/) were greater for MEHP than DEHP in both urine and serum. In contrast, the half-lives (t1/2 [h]) of DEHP were greater than those of MEHP. The AUC ratios between DEHP and MEHP were relatively smaller in serum than in urine, suggesting the important role of urinary DEHP data for exposure assessment of DEHP. The toxicokinetic relationship between DEHP and MEHP in rats suggests that DEHP exposure assessment should be based on DEHP and MEHP in urine and serum for risk assessment applications.     

High-throughput determination of mono- and di(2-ethylhexyl)phthalate migration from PVC tubing to drugs using liquid chromatography-tandem mass spectrometry

The risk assessment of di(2-ethylhexyl) phthalate (DEHP) that migrated from polyvinyl chloride (PVC) medical devices is an important issue for hospitalized patients. Many studies have been conducted to determine the level of DEHP migration. A recent report has indicated that DEHP in blood bags was hydrolyzed by esterase to mono(2-ethylhexyl) phthalate (MEHP). Therefore, a method for the simultaneous determination of DEHP and MEHP was developed. The migration of DEHP and MEHP from PVC tubing to drugs was examined. Although we detected MEHP in the drugs, we found no enzymatic activity involved in the migration process. Some reports have indicated that hydrolysis may have occurred during sterilization by autoclaving. However, we did not perform any heat treatment. It is speculated that the MEHP migrated directly from the PVC tubing. The simultaneous determination of DEHP and MEHP is required for risk assessment, as MEHP may be even more toxic than the parent compound.     

Effect of sterilization process on the formation of mono(2-ethylhexyl)phthalate from di(2-ethylhexyl)phthalate

The risk assessment of di(2-ethylhexyl)phthalate (DEHP) migrating from polyvinyl chloride (PVC) medical devices is an important issue. Many studies have been conducted to determine the level of DEHP migration. A recent report has indicated that DEHP in blood bags is hydrolyzed by esterase into mono(2-ethylhexyl)phthalate (MEHP). However, MEHP is thought to be even more toxic than the parent compound. Therefore, a method for the simultaneous determination of DEHP and MEHP was developed. The limits of quantification (LOQs) of DEHP and MEHP were 2.5 and 0.75 ng/ml, respectively. In this study, the effect of sterilization process on the levels of DEHP and MEHP migration was investigated. The level of migration of DEHP from gamma(gamma)-ray sterilized PVC sheet was low compared with that of the unsterilized control. By contrast, the level of MEHP migration from the gamma-ray sterilized PVC sheet was high compared with that of the unsterilized control. In addition, a high content of MEHP was found in the gamma-ray sterilized PVC sheet.     

Testicular toxicity induced by single dosing of di- and mono-(2-ethylhexyl) phthalate in the rat

The testicular toxicity of di-(2-ethylhexyl) phthalate (DEHP), a widely used plasticizer, and of its major metabolite, mono-(2-ethylhexyl) phthalate (MEHP), was assessed after a single dose in rats. Treatment with a single dose of 2.8 g/kg DEHP or 0.8 g/kg MEHP was sufficient to induce testicular atrophy as observed 7 days after dosing. Such a treatment had no effect on plasma FSH levels, and had varying effects on testicular zinc concentrations. After a single dose of 0.8 g/kg MEHP the testicular toxicity was age-dependent, in that only prepubertal rats were susceptible.     

Mono(2-ethylhexyl)phthalate exhibits genotoxic effects in human lymphocytes and mucosal cells of the upper aerodigestive tract in the comet assay

Phthalic acid esters such as di(2-ethylhexyl)phthalate (DEHP) are widely used as plasticizers in PVC products manufactured for commercial, medical, and consumer purposes. Humans are exposed to phthalates originating, e.g., from blood storage bags, tubing materials, and from food-wrapping. While xenoestrogenic and chronic toxic effects of phthalates have been extensively discussed, there is little data on genotoxic effects in human cells. The alkaline comet assay was used to detect single-strand breaks and alkali labile sites of DNA after incubation of human nasal mucosal cells (n = 11) and peripheral lymphocytes (n = 11) with mono(2-ethylhexyl)phthalate (MEHP), the principal hydrolysis product of DEHP. MEHP showed a dose-dependent enhancement of DNA migration both in human mucosal cells and in lymphocytes. This effect indicates a genotoxic potential of MEHP in human mucosal cells. It confirms previous data obtained on the effect of MEHP on lymphocytes.     

Atropine inhibition of the cardiodepressive effect of mono(2-ethylhexyl)phthalate on human myocardium

Di(2-ethylhexyl)phthalate (DEHP) is a commonly used plasticizer in polyvinylchloride (PVC)-derived plastic. Mono(2-ethylhexyl)phthalate (MEHP), the major metabolite of DEHP, had a reversible, concentration-dependent (15-200 micrograms/ml) negative inotropic effect on a human in vitro atrial trabecular isometric preparation with an IC50 of 85 micrograms/ml. When atropine (22-32 micrograms/ml) was included in the atrial preparation the IC50 was shifted to greater than 120 micrograms/ml, suggesting that MEHP acts in part through the cholinergic receptors.     

Di-(2-ethylhexyl) phthalate is without adjuvant effect in mice on ovalbumin

It has been suggested that one possible contributor to the increasing prevalence of IgE-mediated allergic diseases in Europe and the US is exposure to chemicals that may act as adjuvants. It has been reported previously that certain commonly used phthalate plasticizers, such as di-(2-ethylhexyl) phthalate (DEHP), are able to modify immune responses induced in mice by the common hens' egg allergen ovalbumin (OVA). However, the significance of these observations for human health is unclear, not least because the relevant studies have been conducted exclusively using subcutaneous administration of phthalates. We have therefore investigated the ability of DEHP when applied topically to affect anti-OVA antibody responses induced by subcutaneous exposure to OVA in BALB/c strain mice. Doses of DEHP (50mg) were used that resulted in a marked (approximately 30%) increase in liver weight. Dose-responses were conducted in order to identify doses of OVA that were sub-optimal for both anti-OVA IgG1 and IgE antibody responses: 1microg and 0.05microg, respectively. Under these conditions of exposure, topical administration of DEHP was without impact on antibody responses, regardless of whether DEHP was applied local or distant to the site of OVA immunization. Topical application of concentrations of DEHP that provoked marked systemic effects was without effect on the induction of immune responses.     

Distribution and elimination of di-2-ethylhexyl phthalate (DEHP) and mono-2-ethylhexyl phthalate (MEHP) after a single oral administration of DEHP in rats

The distribution and elimination of di-2-ethylhexyl phthalate (DEHP) and mono-2-ethylhexyl phthalate (MEHP) after a single oral administration of DEHP (25 mmol/kg) were studied. A gas-liquid Chromatographic method was used for the simultaneous determination of MEHP and DEHP. The compounds were extracted with methylene chloride and the monoester was alkylated to the hexyl derivative by solid-liquid phase transfer catalysis in methylethyl ketone. The coefficients of variation of this method for determination of DEHP and MEHP were 8.3% and 11.4% respectively. The concentration of DEHP and MEHP in blood and tissues increased to maximum within 6–24 h after dosing, while the highest levels observed in the heart and lungs occurred within 1 h. At 6 h after administration, the highest ratio of MEHP/DEHP (mol%) were recorded in testes (210%) while the other tissues exhibited less than 100%. MEHP disappeared exponentially with t _1/2 values ranging from 23 to 68 h; DEHP t _1/2 ranged from 8 to 156 h and the t _1/2 values of MEHP in several tissues were slightly longer than DEHP. The t _1/2 values in blood were 23.8 h and 18.6 h for MEHP and DEHP, respectively.     

Effects of gasoline engine emissions on preexisting allergic airway responses in mice

Gasoline-powered vehicle emissions contribute significantly to ambient air pollution. We hypothesized that exposure to gasoline engine emissions (GEE) may exacerbate preexisting allergic airway responses. Male BALB/c mice were sensitized by injection with ovalbumin (OVA) and then received a 10-min aerosolized OVA challenge. Parallel groups were sham-sensitized with saline. Mice were exposed 6 h/day to air (control, C) or GEE containing particulate matter (PM) at low (L), medium (M), or high (H) concentrations, or to the H level with PM removed by filtration (high-filtered, HF). Immediately after GEE exposure mice received another 10-min aerosol OVA challenge (pre-OVA protocol). In a second (post-OVA) protocol, mice were similarly sensitized but only challenged to OVA before air or GEE exposure. Measurements of airway hyperresponsiveness (AHR), bronchoalveolar lavage (BAL), and blood collection were performed approximately 24 h after the last exposure. In both protocols, M, H, and HF GEE exposure significantly decreased BAL neutrophils from nonsensitized mice but had no significant effect on BAL cells from OVA-sensitized mice. In the pre-OVA protocol, GEE exposure increased OVA-specific IgG(1) but had no effect on BAL interleukin (IL)-2, IL-4, IL-13, or interferon (IFN)-gamma in OVA-sensitized mice. Nonsensitized GEE-exposed mice had increased OVA-specific IgG(2a), IgE, and IL-2, but decreased total IgE. In the post-OVA protocol, GEE exposure reduced BAL IL-4, IL-5, and IFN-gamma in nonsensitized mice but had no effect on sensitized mice. These results suggest acute exposure to the gas-vapor phase of GEE suppressed inflammatory cells and cytokines from nonsensitized mice but did not substantially exacerbate allergic responses.     

Simulated neonatal exposure to DEHP and MEHP from PVC enteral nutrition products

The leaching of di(2-ethylhexyl)phthalate (DEHP) and mono(2-ethylhexyl)phthalate (MEHP) from medical products made of polyvinyl-chloride (PVC) to enteral nutrition (EN) for neonatal patients was determined in a simulated study. The study simulated a typical case of EN administration to a neonatal patient (body weight, 3 kg) in a neonatal care unit (temperature, 25 degrees C); the medical products used were an irrigator and catheter containing DEHP (9.1-31.8%, w/w) as a plasticizer. The worst-case daily exposures of the neonatal patient to DEHP and MEHP by the administration of EN were estimated to be 148 and 3.72 microg/(kg day), respectively, as assessed from the levels of these compounds leaching from the medical products to the EN. The use of DEHP-free medical products reduced the exposure of DEHP and MEHP to the minimum levels contained in the EN at preparation. A transition to DEHP-free medical products for neonatal patients would be effective in reducing the exposure of neonatal patients to DEHP via EN administration.