Embryotoxic effects of di-2-ethylhexyl phthalate (DEHP) and di-n-butyl phthalate (DBP) in mice

Two major compounds of PVC plasticizers, di-2-ethylhexyl phthalate (DEHP) and di-n-butyl phthalate (DBP), were mixed with food at levels of 0.05, 0.1, 0.2, 0.4, and 1.0 wt% and given to pregnant mice of ICR-JCL strain throughout gestation. Treatment with 0.2, 0.4, and 1.0% DEHP and 1.0% DBP resulted in decreased maternal weight gain and increased resorption rate. All of the implanted ova died in utero at 0.4 and 1.0% level of DEHP. The malformation rate in term fetuses increased at 0.2% level of DEHP and 1.0% level of DBP, the difference from the control group being at the borderline level of significance. The major malformations observed in the treated groups were neural tube defects (exencephaly and spina bifida), suggesting that the phthalic acid esters could interfere with the closure of neuropores in developing embryos. Treatment with the compounds caused intrauterine growth retardation and delayed ossification with an apparently dose-related response pattern. These results indicate that DEHP and DBP at a high dose level could be embryotoxic and possibly teratogenic mice. The maximum no effect level of the PAEs on mouse fetuses was 70 mg/kg/day, which is far higher than the estimated human current intake from the environment.     

Concentration of di(2-ethylhexyl) phthalate (DEHP) in foods and its dietary exposure in China

Di(2-ethylhexyl) phthalate (DEHP) is a common plasticizer used in food contact materials that has been reported as an endocrine disruptor. In the present study, DEHP concentrations were analyzed in foods in order to determine dietary exposure among the Chinese population, including the general population (aged 2–100) and four age group, that is children aged 2–6, adolescent aged 7–12, young people aged 13–17 and adults aged 18 years old and above. 1704 food samples were collected during 2011–2012 and categorized into 12 food groups which covered major foods in China. Food consumption data were taken from China National Nutrient and Health Survey which was performed in 2002 and includes data from 68,959 subjects. Mean concentrations of DEHP were combined with individual food consumption data to estimate dietary exposure. It was found that DEHP levels in foods ranged from not detected to 3.41 mg/kg, with highest mean values in meat (0.23 mg/kg) and vegetable oils (0.21 mg/kg). Mean dietary intakes of DEHP in the general population, children, and adults were 2.34, 4.51, 2.03 μg/kg bw per day, respectively. The 97.5% intakes in these populations were 5.22, 8.43, 3.64 μg/kg bw per day, respectively. The main food sources of DEHP dietary intake were cereals (39.44%), drinking water (16.94%) and meat (15.81%) in children, and cereals (44.57%), meat (15.70%) and drinking water (12.28%) for adults. These findings suggested that dietary exposure to DEHP among Chinese population was lower than tolerable daily intake of DEHP and there are no health concerns based on generally accepted exposure limits.     

Internal exposure of nursery-school children and their parents and teachers to di(2-ethylhexyl)phthalate (DEHP)

Di(2-ethylhexyl)phthalate (DEHP) is the main plasticizer for polyvinyl chloride (PVC) products. It has become widely spread in our environment and among people. DEHP is suspected to be responsible for endocrine-disruptor-like effects in mankind. Children are probably most susceptible to these endocrine effects. In this study we determined the internal exposure of nursery school children (aged 2-6 years) to DEHP and compared it to their parents' and teachers' exposure. The DEHP-metabolites mono(2-ethyl-5-hydroxyhexyl)phthalate (5OH-MEHP), mono(2-ethyl-5-oxo-hexyl)phthalate (5oxo-MEHP) and mono(2-ethylhexyl)phthalate (MEHP) were determined in first morning urine. The sum of the three DEHP metabolites in children's and in adults' urine was 90.0 and 59.1 micrograms/l respectively (median values; p = 0.074). Concentrations of the secondary metabolites 5OH-MEHP (median: 49.6 vs. 32.1 micrograms/l; p = 0.038) and 5oxo-MEHP (median: 33.8 vs. 19.6 micrograms/l; p = 0.015) were significantly higher in children than in adults. MEHP concentrations were low both in adults and children (median: 6.6 micrograms/l vs. 9.0 micrograms/l). Creatinine adjusted values should more accurately reflect the dose taken up with respect to body weight when comparing children with adults. Total creatinine adjusted DEHP metabolites in urine were significantly higher in children than in adults (median values: 98.8 vs. 50.9 micrograms/g creatinine; p < 0.0001). This also applied to the concentrations of both secondary metabolites 5OH-MEHP (55.8 vs. 28.1 micrograms/g creatinine; p < 0.0001) and 5oxo-MEHP (38.3 vs. 17.2 micrograms/g creatinine; p < 0.0001). Creatinine corrected concentrations for the monoester MEHP in children and adults were very similar (8.7 vs. 8.6 micrograms/g creatinine; p = 0.908). Based on the sum of the three determined metabolites we estimated the DEHP dose (in microgram/kg body-weight) taken up by children to be about twice as high as the dose taken up by adults. Routes of the ubiquitous exposure to DEHP remain indistinct. In children's urine the mean relative ratios of MEHP to 5OH-MEHP to 5oxo-MEHP were 1 to 7.1 to 4.9, in adults they were 1 to 3.4 to 2.1. This might indicate an enhanced oxidative metabolism in children. To date no information on the biological activity and toxicity of oxidative metabolites of DEHP is available. Since these are the major metabolites of DEHP toxicological data on these metabolites is urgently needed.     

Effect of di-2-ethylhexyl phthalate (DEHP) on rat gonads.

Activity of succinic dehydrogenase and adenosine triphosphatase was significantly reduced and that of β-glucuronidase was increased in rat gonads after treatment with di-2-ethylhexyl phthalate (DEHP). Histopathological studies revealed focal degeneration of seminiferous tubules and edema of interstitium in testis but no detectable alterations in ovary of treated animals as compared to controls.Such alterations may perhaps be responsible for the reported reproductive dysfunction in experimental animals after exposure to this and other phthalate plasticizers.     

邻苯二甲酸二(2-乙基己基)酯对青春前期雄性大鼠的生殖毒性

目的探讨邻苯二甲酸二(2-乙基己)酯(di-2-ethylhexyl phthalate,DEHP)对青春前期雄性大鼠的生殖毒性。方法将32只健康21日龄清洁级雄性SD大鼠按体重随机分为4组,分别为溶剂对照(玉米油)组和250、500、1 000 mg/kg DEHP染毒组,每组8只。采用灌胃方式进行染毒,染毒容量为10 ml/kg,每天1次,连续染毒4周。采用CASA精子分析系统分析精子活力相关指标。结果与对照组比较,1 000 mg/kg DEHP染毒组大鼠的精子密度、精子活动率、活动精子密度、最大侧摆幅度、直线运动的精子密度及A级精子百分率均较低,而D级精子百分率较高;250 mg/kg DEHP染毒组平均直线运动速度、精子运动的向前性均较低;500、1 000 mg/kg DEHP染毒组直线运动的精子个数均较低,差异均有统计学意义(P0.05);而各剂量DEHP染毒组B、C级精子比例和平均曲线运动速度、平均路径速度、平均侧摆幅度、平均鞭打频率、运动的直线性、运动的摆动性、平均移动角度、直线运动精子活率均无明显改变。结论 DEHP对青春前期雄性大鼠精子活力产生明显的毒性作用,从而影响雄性的生殖功能。     

Effects of di(2-ethylhexyl) phthalate and dibutyl phthalate on the collembolan Folsomia fimetaria

Lethal and sublethal effects of di(2-ethylhexyl) phthalate (DEHP) and dibutyl phthalate (DBP) on adult individuals of the collembolan Folsomia fimetaria were investigated in the laboratory by the use of small microcosms. Effects of DEHP and DBP were also tested on newly hatched collembolans in a multidish system. The endpoints were juvenile mortality, growth, and development. When exposed to DEHP, adults and juveniles were unaffected at all test concentrations, that is, up to 5,000 mg/kg. However, DBP caused increased adult mortality at 250 mg/kg and juvenile mortality at 25 mg/kg. For DBP, adult reproduction was a more sensitive endpoint than was survival, with an EC10 and EC50 of 14 and 68 mg/kg, respectively. Juvenile molting frequency seems to be a sensitive parameter, because number of cuticles produced by young springtails was reduced at 1 mg/kg. Toxicity was reduced when soil spiked with DBP was stored at 20 degrees C for a period of up to 28 d before adding the animals. Reduction in toxicity of DBP may be due a combination of degradation, evaporation, and adsorption of DBP to soil material. This was confirmed by chemical analyses, which showed a rapid initial disappearance followed by a much slower disappearance. Our results lead to the overall conclusion that significant adverse effects of phthalates on collembolans are not likely to occur as a result of normal sewage sludge application.     

Daily intake of di(2-ethylhexyl)phthalate (DEHP) by German children -- A comparison of two estimation models based on urinary DEHP metabolite levels

Di(2-ethylhexyl)phthalate (DEHP) is a general-purpose plasticizer for polyvinyl chloride (PVC) and has become a ubiquitous environmental contaminant. It is suspected to be an endocrine disrupting/modulating substance in humans. Children are of special concern due to their developmental state. In our study we estimated the daily DEHP intake of 239 children aged 2-14 years by extrapolating from their urinary levels of the DEHP metabolites mono-(2-ethyl-5-hydroxyhexyl)phthalate (5OH-MEHP), mono-(2-ethyl-5-oxohexyl)phthalate (5oxo-MEHP) and mono-(2-ethylhexyl)phthalate (MEHP). We applied two calculation models based upon the volume and the creatinine-related urinary metabolite concentrations. Applying the volume- or the creatinine-based calculation model we determined a median daily DEHP intake of 7.8 or 4.3 microg/kgbody weight (bw)/day and a 95th percentile of 25.2 or 15.2 microg/kgbw/day. Three children (1%) exceeded the value of the tolerable daily intake (TDI) of the European Food Safety Authority of 50 microg/kgbw/day, while 7.5% or 3% (depending on the calculation model) exceeded the reference dose (RfD) of 20 microg/kgbw/day of the US Environmental Protection Agency. In general, DEHP exposure was decreasing with increasing age and boys had higher exposures than girls. Our findings suggest that the majority of the children in the general population is exposed to quantities of DEHP below the TDI and the RfD. However, many children scoop out the preventive limit values to a considerable degree and in individual cases we observed substantial transgressions. Younger children seem to be more severely burdened, which may be due to a higher food consumption related to their bw, mouthing behaviour and/or playing near the ground.     

Species and inter-individual differences in metabolic capacity of di(2-ethylhexyl)phthalate (DEHP) between human and mouse livers

Objectives

This study was conducted to assess inter-species and inter-individual differences in the metabolism of di(2-ethylhexyl)phthalate (DEHP) in humans and mice.

Methods

The activities of four DEHP-metabolizing enzymes [lipase, UDP-glucuronocyltransferase (UGT), alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH)] were measured in the livers of 38 human subjects of various ages and in eight 129/Sv male mice.

Results

Microsomal lipase activity was significantly lower in humans than in mice. The V _max/K _m value in humans was one-seventh of that in mice, microsomal UGT activity in humans was a sixth of that in mice, and cytosolic ALDH activity for 2-ethylhexanal in humans was one-half of that in mice. In contrast, ADH activity for 2-ethylhexanol was twofold higher in humans than in mice. The total amount of DEHP urinary metabolites and the concentration of mono(2-ethylhexyl)phthalate (MEHP) were much higher in intact mice than in the U.S. general population based on data reported elsewhere, regardless of the similar estimated DEHP intake between these mice and the human reference population. However, mono(2-ethyl-5-oxo-hexyl)phthalate (5oxo-MEHP) and mono(2-ethyl-5-carboxypentyl)phthalate (5cx-MEPP) levels were higher in the latter than in the former. Of note, inter-subject variability in the activities of all enzymes measured was 10–26-fold.

Conclusion

The inter-individual variation in the metabolism of DEHP in humans may be greater than the difference between mice and humans (inter-species variation), and both may affects the risk assessment of DEHP.     

Studies by the National Toxicology Program on di(2-ethylhexyl)phthalate

In a 2-year feed study previously reported by the National Toxicology Program (NTP), the plasticizer di(2-ethylhexyl)phthalate (DEHP) was found to produce increased incidences of hepatocellular neoplasms in both sexes of Fischer 344 (F344) rats and B6C3F1 mice. Further studies by the NTP on this chemical have investigated its genotoxicity, dermal absorption, reproductive and developmental toxicity, and biochemical mechanism of action. DEHP was not mutagenic in Salmonella typhimurium (strains TA98, TA100, TA1535 or TA1537), in L5178Y mouse lymphoma cells, or in Drosophila melanogaster. DEHP did not induce chromosomal aberrations, but did cause a marginal dose-related increase in sister chromatid exchanges in CHO cells. In a dermal absorption study, DEHP was not absorbed well through the skin of F344 rats. In a fertility assessment study, DEHP was shown to be a reproductive toxicant in both male and female CD-1 mice. The teratogenic potential of DEHP was evaluated in F344 rats and CD-1 mice. In the rat study, there were no significant differences in percent fetuses malformed between control and treatment groups, even at dose levels (1.0, 1.5 and 2.0%) which produced significant maternal and fetal toxicity. In the mouse study, the incidence of fetuses with malformations was significantly increased at dose levels which produced maternal and/or fetal toxicity (0.10 and 0.15%), and at a dose level (0.05%) which did not cause maternal or fetal toxicity. The no-observed effect level for developmental toxicity in mice was 0.025% DEHP. Kinetic data on the rates of formation of H2O2 by peroxisomal palmitoyl CoA oxidase, and of degradation of H2O2 by catalase, was used to estimate in vitro steady-state H2O2 concentrations during peroxisomal oxidation of palmitoyl CoA. Increases in steady-state H2O2 in liver homogenates of rats treated with DEHP, di(2-ethylhexyl)adipate, or nafenopin, a hypolipidemic drug, correlated well with the carcinogenic potential of these chemicals determined in previous carcinogenicity studies, and are consistent with but not definitive evidence for the involvement of peroxisome proliferation in the hepatocarcinogenesis of these compounds.     

Follow-up study of adolescents exposed to di(2-ethylhexyl) phthalate (DEHP) as neonates on extracorporeal membrane oxygenation (ECMO) support

Di(2-ethylhexyl) phthalate (DEHP) is used to make polyvinyl chloride (PVC) plastic tubing soft and flexible. Animal data show that adverse effects of DEHP exposure may include reduced fertility, reduced sperm production in males, and ovarian dysfunction in females. Known treatments that involve high DEHP exposures are blood exchange transfusions, extracorporeal membrane oxygenation (ECMO), and cardiovascular surgery. Although potential exposure to DEHP in ECMO patients is significant, the exposure has not been associated with short-term toxicity. To evaluate long-term toxicity, we undertook a study of neonatal ECMO survivors to assess their onset of puberty and sexual maturity. We evaluated 13 male and 6 female subjects at 14-16 years of age who had undergone ECMO as neonates. All subjects had a complete physical examination including measurements for height, weight, head circumference, and pubertal assessment by Tanner staging. The testicular volume and the phallic length were measured in male participants. Laboratory tests included thyroid, liver, and renal function as well as measurements of luteinizing hormone, follicle-stimulating hormone, testosterone for males, and estradiol for females. Except for one patient with Marfan syndrome, the rest had normal growth percentile for age and sex. All had normal values for thyroid, liver, and renal functions. Sexual hormones were appropriate for the stage of pubertal maturity. Our results indicate that adolescents exposed to significant quantities of DEHP as neonates showed no significant adverse effects on their physical growth and pubertal maturity. Thyroid, liver, renal, and male and female gonadal functions tested were within normal range for age and sex distribution. Key Words: DEHP, ECMO, toxicity.     

An estimation of the daily intake of di(2-ethylhexyl)phthalate (DEHP) and other phthalates in the general population.

We analyzed 85 urine samples of the general German population for human specific metabolites of phthalates. By that we avoided contamination with the parent phthalates being omnipresent in the environment and for the first time could deduce each individual's internal exposure to phthalates without contamination. Determined were the secondary metabolites mono(2-ethyl-5-hydroxyhexyl)phthalate (5OH-MEHP) and mono(2-ethyl-5-oxo-hexyl)phthalate (5oxo-MEHP) of di(2-ethylhexyl)phthalate (DEHP) and the primary monoester metabolites of DEHP, di-noctylphthalate (DnOP), di-n-butylphthalate (DnBP), butylbenzylphthalate (BBzP) and diethylphthalate (DEP). Based on these internal exposure values we calculated the daily intake of the parent phthalates using urinary metabolite excretion factors. For DEHP we determined a median intake of 13.8 micrograms/kg body weight/day and an intake at the 95th percentile of 52.1 micrograms/kg body weight/day. The tolerable daily intake (TDI) value settled by the EU Scientific Committee for Toxicity, Ecotoxicity and the Environment (CSTEE) is 37 micrograms/kg body weight/day. Twelve percent of the subjects (10 out of 85 samples) within our collective of the general population are exceeding this value. Thirty-one percent of the subjects (26 out of 85 samples) had values higher than the reference dose (RfD) of 20 micrograms/kg body weight/day of the U.S. Environmental Protection Agency (EPA). For DnBP, BBzP, DEP and DnOP intake values at the 95th percentile were 16.2, 2.5, 22.1 and 0.42 micrograms/kg body weight/day respectively. Our results unequivocally prove that the general German population is exposed to DEHP to a much higher extent than previously believed. This is of greatest importance for public health since DEHP is not only the most important phthalate with respect to its production, use, occurrence and omnipresence but also the phthalate with the greatest endocrine disrupting potency. DEHP is strongly suspected to be a developmental and reproductive toxicant. We are not aware of any other environmental contaminant for which the TDI and RfD are exceeded to such an extent within the general population. The transgressions of the TDI and RfD for DEHP are accompanied by considerable ubiquitous exposures to DnBP and BbzP, two phthalates under scrutiny for similar toxicological mechanisms.     

邻苯二甲酸二-(2-乙基己基)酯对小鼠胚胎发育毒性的体外实验研究

为探讨邻苯二甲酸 二 2 乙基己基酯 (Di 2 ethylhexylphthalate ,DEHP)对体外培养小鼠胚胎的发育毒性 ,采用植入后全胚胎培养模型 ,将 8.5天龄Balb c小鼠胚胎移入含DEHP即刻离心血清培养 48h ,DEHP终浓度为 0、12 .5、2 5、5 0、10 0和 2 0 0mg L ,观察DEHP对小鼠胚胎生长发育和组织器官形态分化的影响。结果显示DEHP对体外培养小鼠胚胎生长发育毒性的最大无作用剂量为 12 .5mg L ,≥ 2 5mg L的DEHP可诱发胚胎生长迟缓及组织器官形态分化异常 ,出现心脏、神经系统、腮弓发育异常及小肢芽、体位异常等畸形 ;10 0mg L的DEHP对体外培养胚胎偶尔呈致死效应 ;2 0 0mg L时各组织器官形态分化均出现了异常 ,但未发现死胎。上述改变具有明显的剂量 -效应及剂量 -反应关系。提示DEHP对体外培养的小鼠胚胎具有胚胎毒性 ,并以致畸效应为主     

In vivo studies on the mechanism of di(2-ethylhexyl)phthalate carcinogenesis

In a study sponsored by the National Toxicology Program, di(2-ethylhexyl)phthalate (DEHP) fed in the diet at 1.2% significantly increased the incidence of female rats with hepatocellular carcinomas. Extensive evaluation of DEHP for carcinogenicity has yielded negative results. The present investigations were designed to elucidate the mechanism of DEHP hepatocarcinogenesis under the conditions of the original bioassay. Short-term studies designed to evaluate the promoting capability of DEHP, when administered after initiation, were negative when livers of female Fischer-344 rats were evaluated using multiple histochemical stains to identify foci of cellular alteration. Two different protocols were used to evaluate the initiating potential of DEHP in the liver using histochemically defined foci as the endpoint. In both experiments the results were negative. Chronic exposure to DEHP at 1.2% in the diet for 2 years resulted in elevation of hepatic peroxisomal enzymes while DNA replication, an indication of cell proliferation, was not affected in hepatocytes. The number of foci was not elevated in the DEHP group compared to the controls, even though a low incidence of rats with liver tumors occurred in the treated group. The results of this series, as well as other published results, suggest that DEHP and other peroxisomal proliferating chemicals have unique effects on the development of hepatic neoplasms. The absence of altered foci after chronic administration or in initiation-promotion protocols distinguishes DEHP and perhaps other peroxisomal proliferating chemicals from both classic liver carcinogens and promoters.     

邻苯二甲酸二(2-乙基)己基酯影响白细胞介素-4蛋白的机制初探

目的 通过研究钙调神经磷酸酶(CaN)在邻苯二甲酸二(2-乙基)己基酯(DEHP)影响白细胞介素(IL)-4蛋白表达中的作用,初步探讨DEHP的免疫毒性机制.方法 以10μg/T佛波酯(PMA)及0.5 mg/L离子霉素(Ion)为激活剂,用10、50 μmol/L DEHP染毒脾淋巴细胞72及96 h,同时用0.5 μmol/L CaN抑制剂FK506进行干预.实验终点检测细胞上清液中乳酸脱氢酶(LDH)漏出、用酶联免疫吸附试验测定细胞上清液中CaN蛋白及IL-4蛋白含量.结果 与PMA+Ion组比较,10、50 μmol/L DEHP均使细胞上清液中LDH漏出明显升高;50 μmol/L DEHP作用细胞72及96 h,使CaN蛋白表达分别增加3.83倍及3.91倍.实验72 h,50 μmol/L DEHP与PMA+Ion组比较明显促进IL-4蛋白表达,且FK506能抑制淋巴细胞分泌IL-4蛋白.结论 CaN在DEHP调控IL-4蛋白表达中具有重要作用.     

青春期前邻苯二甲酸二(2-乙基)己酯暴露对雌性大鼠生殖发育及过氧化物酶体增殖剂激活受体的影响

目的探讨邻苯二甲酸二(2-乙基)己酯(DEHP)暴露对青春期前雌性大鼠性发育及生殖内分泌功能的影响及其可能机制。方法将40只健康3周龄雌性SD大鼠,随机分成对照组(玉米油)和3个实验组,每组10只。实验组按50、150、500mg/kg DEHP经口灌胃,连续染毒28天。观察阴道开口、乳房发育、第一次发情周期的日龄及体重,于末次染毒24小时后进行阴道涂片,确定动情间期,处死动物。Real-time PCR测定卵巢组织相关基因表达水平;ELISA法检测血清中卵泡刺激素(FSH)、黄体生成素(LH)、雌二醇(E2)、孕酮(P4)及睾酮(T)水平;通过病理学观察卵巢组织的变化;免疫组织化学法测定卵巢组织中PPARγ的表达。结果 500mg/kg组阴道开口日龄提前,150、500mg/kg组阴道开口时体重增加(P<0.05);150、500mg/kg组芳香化酶(P450Arom)mRNA表达与对照组比较,显著下降;各剂量组与对照组相比,T水平明显下降,150、500mg/kg组FSH、E2水平明显减少,而LH水平明显升高(P<0.05);150、500mg/kg组闭锁卵泡明显增多、黄体数目明显减少;150、500mg/kg组卵泡颗粒细胞和黄体颗粒细胞中PPARγ阳性光密度相对量明显高于对照组及50mg/kg组(P<0.05)。结论青春期前DEHP暴露对雌性大鼠性发育及生殖内分泌功能产生影响,其作用机制可能与激活PPARs有关。     

Inhibition of alveolar macrophage killing by di(2-ethylhexyl)phthalate

The phthalate ester plasticizer di(2-ethylhexyl) phthalate (DEHP) affected the phagocytosis and killing ofStaphylococcus aureus by rabbit alveolar macrophages. Pre-exposure to DEHP caused a slight increase in phagocytosis and a dose-dependent inhibition of alveolar macrophage killing ofS. aureus, reaching a 14-fold decrease in the killing index at a concentration of 2 mg DEHP per 100 ml of media. The significance of this inhibition is discussed with respect to the role of the plasticizer in lung disease and macrophage research.     

Di(2-ethylhexyl)phthalate (DEHP) exposure of voluntary plasma and platelet donors

Each year thousands of healthy volunteers undergo apheresis procedures to donate blood components and safe lives. However, many disposables used in apheresis contain di(2-ethylhexyl)phthalate (DEHP). This way, donors are exposed to DEHP, which is a reproductive and developmental toxicant in animals and a suspected endocrine modulator in humans. We quantified the DEHP exposure of six plasma donors, six discontinuous-flow platelet donors and six continuous-flow platelet donors by determining three specific metabolites in urine (5OH-MEHP: mono(2-ethyl-5-hydroxyhexyl)phthalate; 5oxo-MEHP: mono(2-ethyl-5-oxo-hexyl)phthalate and MEHP: mono(2-ethylhexyl)phthalate). We found maximum concentrations in urine samples after the discontinuous-flow plateletpheresis procedure with 826 microg/l for 5OH-MEHP, 774 microg/l for 5oxo-MEHP and 266 microg/l for MEHP (mean of the six volunteers). Metabolite excretions were found to be significantly (p<0.0001) higher for both plateletpheresis techniques compared to plasmapheresis and controls. Continuous-flow plateletpheresis led to significantly higher (p<0.0001) excretions than discontinuous-flow plateletpheresis. Mean absolute DEHP exposures were 1.2 mg for discontinuous- and 2.1 mg for continuous-flow plateletpheresis. Exposure for plasmapheresis (0.37 mg) was in the range of the controls (0.41 mg). Mean DEHP doses for both plateletpheresis techniques (18.1 and 32.3 microg/kg/day) were close to or exceeded the reference dose (RfD) of the US EPA and tolerable daily intake (TDI) value of the EU on the day of the apheresis. Therefore, margins of safety might be insufficient to protect especially young men and women in their reproductive age from effects on reproductivity. At present, discontinuous-flow devices should be preferred to avert conceivable health risks from plateletpheresis donors. Strategies to avoid DEHP exposure of donors during apheresis need to be developed.