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international journal of andrology ISSN 0105-6263

REVIEW ARTICLE

Phthalates: metabolism and exposure Matthias Wittassek and Ju¨rgen Angerer Institute and Outpatient Clinic of Occupational, Social and Environmental Medicine, University of Erlangen-Nuremberg, Erlangen, Germany

Summary Keywords: biomonitoring, cumulative TDI, human exposure, human metabolism, phthalates Correspondence: Ju¨rgen Angerer, Institute and Outpatient Clinic of Occupational, Social and Environmental Medicine, University of Erlangen-Nuremberg, Schillerstr. 25 ⁄ 29, D-91054 Erlangen, Germany. E-mail: [email protected] Received 18 July 2007; revised 9 September 2007; accepted 11 October 2007 doi:10.1111/j.1365-2605.2007.00837.x

In human metabolism studies we found that after oral application of di(2-ethylhexyl) phthalate (DEHP), diisononyl phthalate (DiNP) and di(2-propylheptyl) phthalate (DPHP), at least 74, 44 and 34%, respectively, are excreted via urine. In contrast to the short chain phthalates, their oxidized products, not the simple monoesters, were found to be the main metabolites. Based on urinary phthalate metabolite concentrations we estimated in 102 German subjects between 6 and 80 years of age median daily intakes (lg ⁄ kg ⁄ day) of 2.7 for DEHP, 2.1 for di-nbutyl phthalate, 1.5 for diisobutyl phthalate, 0.6 for DiNP, and 0.3 for butylbenzyl phthalate. In general, children have higher exposures compared to adults and seem to have a more effective oxidative metabolism of phthalates. For individual phthalates tolerable daily intake (TDI) values have been deduced. However, in rats some phthalates have been shown to act as endocrine disrupters via a common mechanism of action in a dose-additive manner. Therefore, the concept of a cumulative TDI value may be more appropriate for the consideration of the overall exposure and the potential human health risks resulting from everyday and simultaneous exposure to several phthalates.

Introduction Phthalates are mostly used as plasticizers in PVC products. Annually more than 1 000 000 tonnes of phthalates are consumed in Western Europe (AgPU, 2006). Among the most important phthalates are di-n-butyl phthalate (DnBP), diisobutyl phthalate (DiBP), butylbenzyl phthalate (BBzP), di(2-ethylhexyl) phthalate (DEHP), diisononyl phthalate (DiNP), diisodecyl phthalate (DiDP) and di(2-propylheptyl) phthalate (DPHP). As ubiquitous environmental contaminants phthalates are taken up by the general population. This has raised concern because phthalates are considered to be endocrine-disrupting chemicals. It is supposed that phthalates and other endocrine-disrupting chemicals might have contributed to adverse trends in reproductive medicine, which show an increase in testicular cancer and hypospadias as well as a decrease in sperm counts (Sharpe & Irvine, 2004). Human phthalate metabolism: single oral dose experiments Di(2-ethylhexyl) phthalate For many years DEHP was the most important PVC plasticizer. Therefore, we decided to study intensively human metabolism of DEHP. For this purpose a healthy male

volunteer at the age of 61 took three different oral doses of isotopically labelled DEHP – about 5, 29 and 650 lg ⁄ kg body weight (Koch et al., 2004a, 2005a). By using D4-ring-labelled DEHP interference by the omnipresent background exposure to DEHP was avoided. After 24 h the urinary excretion rate for the sum of five metabolites, namely mono-(2-ethylhexyl) phthalate (MEHP), mono-(2-ethyl-5-hydroxyhexyl) phthalate (5OH-MEHP), mono-(2-ethyl-5-oxohexyl) phthalate (5oxo-MEHP), mono(2-ethyl-5-carboxypentyl) phthalate (5cx-MEPP), and mono[2-(carboxymethyl)hexyl] phthalate (2cxMMHP), was between 65 and 70% for all three doses. This outcome was in contrast to previous studies, which had hypothesized that a great amount of DEHP is excreted with faeces (Schmid & Schlatter, 1985). After 2 days more than 65% of the highest dose1 taken up were excreted as the sum of the four oxidative metabolites, whereas only 7.3% was excreted as MEHP (Table 1). We found that urinary DEHP excretion follows a multi-phase elimination model. In the second elimination phase MEHP showed the shortest half-life with 5 h, whereas for 2cx-MMHP a half-life of around 24 h was estimated. Interestingly, there were two maximum concentrations 1

For the medium- and low-dose cases, only 24 h urine samples were collected.

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for 2cx-MMHP (after 9 and 24 h), which appeared much later than those of the other metabolites (2–4 h). In parallel with the measurements in urine we also detected all five metabolites in blood serum samples of the volunteer (Koch et al., 2004a, 2005a). However, in blood serum the simple monoester MEHP was clearly the dominating metabolite. In the high-dose experiment a peak value of 4.95 mg ⁄ L was measured 2 h after oral application. The half-lives were around 2 h for all metabolites except for 2cx-MMHP with a half-life of at least 5 h. After normalization by the dose we calculated a 15– 100 times higher normalized area under the concentration-time curve for MEHP in the blood of the human volunteer than previously found in rats and marmosets (Kessler et al., 2004). This may indicate that a similar external exposure to DEHP results in a higher internal dose to MEHP in humans compared to rats and marmosets. This issue deserves further investigation as it may be essential when considering dose–effect relationships in humans and animals. Diisononyl phthalate and di(2-propylheptyl) phthalate After investigating the metabolism of DEHP we turned towards the metabolism of DiNP. In Europe DiNP together with DiDP, both mixtures of variously branched isononyl and isodecyl isomers, respectively, have replaced DEHP as the major plasticizer for PVC products in recent years (AgPU, 2006). A healthy male volunteer, aged 63, took a single oral dose of 1.27 mg ⁄ kg body weight of the deuterium analogue of the commercial product DiNP 2 (Koch & Angerer, 2007). We assumed that DiNP would be metabolized in the same way as DEHP by hydrolysis and subsequent x and x-1 oxidation. However, as DiNP is a mixture of various alkyl isomers, not only one monoester, one hydroxy, one oxo and one carboxy metabolite, but a variety of isomeric metabolites is formed and excreted in urine. Therefore, we determined the sum of the simple monoesters and the sum of the monoesters with one hydroxy (OH-MiNP), one oxo (oxo-MiNP) and one carboxy group (cx-MiNP), respectively. Within 49 h we recovered 43.6% of the applied DiNP dose in the urine of the volunteer. The highest share was found for the hydroxy compounds accounting for 20.2% of the dose, followed by the oxo and carboxy products with each around 11%. The monoester was merely a minor metabolite with 2.2%. As in the case of DEHP elimination of DiNP followed a multi-phase pattern, with the second elimination phase beginning 24 h post-dose. Within this phase we estimated half-lives between 5 h for MiNP and 18 h for the carboxy products (Table 1). The maximum concentrations were measured between 2 h for MiNP and 8 h for cx-MiNP after application. 132

Table 1 Renal excretion of metabolites in percentage of the oral dose after a single oral dose of D4-DEHP (650 lg ⁄ kg BW) and D4DiNP (1.27 mg ⁄ kg BW), respectively, maximal concentrations, times of the maximal concentration, and estimated half-lives for the first and second elimination phase (Koch et al., 2004a, 2005a; Koch and Angerer, 2007) Urinary excretion cmax (%) (mg ⁄ L) DEHP

44 h pd

MEHP 7.3 5OH-MEHP 24.7 5oxo-MEHP 14.9 5cx-MEPP 21.9 2cx-MMHP 5.4

DiNP MiNP OH-MiNP oxo-MiNP cx-MiNP

tmax (h)

First phase

Second phase

t½ (h)

t½ (h)

8–16 h pd 16–24 h pd 3.6 2 10.0 4 6.2 4 8.2 4 0.9 and 9 and 1.2 24

49 h pd 2.2 20.2 10.6 10.7

2.6 22.4 11.1 10.9

2 4 4 8

2 2 2 3 3

5 10 10 12–15 24

8–24 h pd

24 h pd

3 5 5 5

5 12 12 18

Pd, post-dose; DEHP, di(2-ethylhexyl) phthalate; 5OH-MEHP, mono-(2ethyl-5-hydroxyhexyl) phthalate; 5oxo-MEHP, mono-(2-ethyl-5-oxohexyl) phthalate; 5cx-MEPP, mono(2-ethyl-5-carboxypentyl) phthalate; 2cx-MMHP, mono[2-(carboxymethyl)hexyl] phthalate; DiNP, diisononyl phthalate; OH-MiNP, monoisononyl phthalates with one hydroxy group; Oxo-MiNP, monoisononyl phthalates with one oxo group; cx-MiNP, monoisononyl phthalates with one carboxy group.

Recently, we investigated the human metabolism of DPHP. First results indicate that after 61 h around 34% of the applied dose is excreted in urine as hydroxy, oxo and carboxy metabolites (Fig. 1). The simple monoester seems to be even lower than in the DiNP experiment representing less than 1% of the dose. Phthalate exposure of the general population Several biomonitoring studies have determined the internal phthalate exposure of the general population by measuring urinary concentrations of phthalate metabolites (Blount et al., 2000; Koch et al., 2003; Silva et al., 2004; CDC, 2005; Wittassek et al., 2007b). In a recent study we determined metabolites of DnBP, DiBP, BBzP, DEHP, and DiNP in a group of 102 persons between 6 and 80 years of age living in south Germany (Koch, H.M., Wittassek, M., Spickenheuer, A., Mu¨ller, J. and Angerer, J., unpublished data). Metabolites of all five phthalates were detectable in virtually each urine sample. Median concentrations (lg ⁄ L) were 50.4 for mono-n-butyl phthalate, 35.7 for monoisobutyl phthalate, 5.4 for monobenzyl phthalate, 4.1 for MEHP, 13.8 for 5OH-MEHP, 12.2 for

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ª 2008 The Authors International Journal of Andrology 31, 131–138

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Phthalates: metabolism and exposure

Figure 1 Renal excretion of phthalate metabolites in relation to oral doses (Anderson et al., 2001; Koch et al., 2005a; Koch & Angerer, 2007). cx-MEHP represents the sum of 5cx-MEPP and 2cx-MMHP. OH-MiNP, oxo-MiNP, cx-MiNP and OH-MPHP, oxo-MPHP and cxMPHP represent the sum of all isomers with the respective functional group in the alkyl side chains.

5oxo-MEHP, 21.6 for 5cx-MEPP, 6.3 for 2cx-MMHP, 2.0 for OH-MiNP, 1.3 for oxo-MiNP and 4.0 for cx-MiNP. By comparing the results with the data of the U.S. National Health and Nutrition Examination Survey (NHANES) 2001 ⁄ 2002 (CDC, 2005) we see very similar median concentrations for the metabolites of DEHP in both countries. But, in the USA the concentrations of the butyl phthalate metabolites were lower, whereas the values for monobenzyl phthalate were higher (medians in lg ⁄ L: MnBP 20.4; MiBP 2.6; MBzP 15.7). Possibly different patterns of use in Germany and the USA may lead to different phthalate exposures. Based on the urinary metabolite concentrations and applying urinary excretion factors determined in human metabolism studies (Anderson et al., 2001; Koch et al., 2005a; Koch & Angerer, 2007) (Fig. 1) we also estimated the amounts of phthalates taken up by the 102 subjects. Highest intakes were calculated for DEHP, DnBP, and DiBP with median (maximal) values of 2.7 (42.2), 2.1 (230), and 1.5 (27.3) lg ⁄ kg bodyweight and day, respectively. For DiNP and BBzP the values were somewhat lower with 0.6 (36.8) and 0.3 (2.2) lg ⁄ kg ⁄ day, respectively. The Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food (AFC) of the European Food Safety Authority (EFSA) has allocated tolerable daily intake (TDI) values for some phthalates (EFSA, 2005a,b,c,d,e). Among the 102 individuals investigated three had a daily DnBP intake above the TDI value (10 lg ⁄ kg ⁄ day). However, an isolated view on each phthalate does not account for the fact that there is simultaneous exposure to several phthalates, all of which may act in a similar way. In animals additive effects of the phthalates have clearly been shown (Borch et al., 2004; Gray et al., 2006; Howdeshell et al., 2007). Howdeª 2008 The Authors Journal compilation ª 2008 Blackwell Publishing Ltd



shell et al. (2007) found that coadministration of DnBP and DEHP in rats during sexual differentiation increases the incidence of a number of reproductive malformations (e.g. epididymal agenesis, reduced androgen-dependent organ weights) in a cumulative, dose-additive manner by suppressing testicular androgen synthesis and insulin-like peptide 3 (insl3) expression. In human risk assessment, these additive effects can be taken into account by summing up the exposure to the individual phthalates that act in a similar mode of action in consideration of their relative toxicity. One way to do this could be to divide the daily exposure of these phthalates by their corresponding TDI values based on reproductive effects and to add up the obtained percentages. A sum value of 100% can then be regarded as a cumulative TDI value, which is not to be exceeded. This approach is in some contrast to a statement given in 2005 by the AFC panel, which said that a group-TDI for BBzP, DBP, DEHP, DiNP and DiDP cannot be allocated (EFSA, 2005f). This was justified by the fact that the TDI values of DiNP and DiDP are based on adverse effects on the liver and not on reproductive toxicity and by the assumption that for DnBP, BBzP and DEHP the profile of the reproductive effects – the TDI values of these phthalates are all based on testicular toxicity – may not be identical at the hormonal and cellular levels. However, for DnBP (10 lg ⁄ kg ⁄ day) and DEHP (50 lg ⁄ kg ⁄ day) both TDI values were deduced on adverse effects on germ cells in rats (EFSA, 2005a,b), based on a LOAEL of 2 mg ⁄ kg ⁄ day (Lee et al., 2004) and a NOAEL of 5 mg ⁄ kg ⁄ day (Wolfe & Layton, 2003), respectively. Due to their similar mode of action, the recently and clearly demonstrated dose-additive effects on the reproductive system of male rats (Howdeshell et al., 2007), and their relatively high exposures in the general population, we think that it is justified to follow an approach of a cumulative TDI for DnBP and DEHP. Therefore, we considered exposure to DnBP and DEHP in the 102 investigated people with respect to a cumulative TDI value for these phthalates (Fig. 2). There were four subjects with phthalate exposure above the cumulative TDI value (max 2300%). However, one has to bear in mind that, next to DnBP and DEHP, there are further phthalates showing reproductive toxicity like BBzP (EFSA, 2005d), DiBP (Borch et al., 2006; Saillenfait et al., 2006) and DiNP (Gray et al., 2000). How far the TDI values of further phthalates should be implemented in the concept of a cumulative TDI value is to be discussed in future (EFSA, 2005f). Phthalate exposure and metabolism in children Children are of special concern with respect to phthalate exposure because of their developmental state. In

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Figure 2 Relative cumulative frequencies of daily intake in relation to the cumulative tolerable daily intake (TDI) of di-n-butyl phthalate (DnBP) and di(2-ethylhexyl) phthalate (DEHP) determined in 102 German subjects between 6 and 80 years of age and in 239 children between 2 and 14 years of age (creatinine-based calculation model) (Koch et al., 2007; Wittassek et al., 2007a). Four of the 102 subjects and 43 children showed exposure to phthalates above the cumulative TDI value for DnBP and DEHP (100%).

cooperation with the German Federal Environmental Agency we investigated the internal phthalate exposure of 239 children between 2 and 14 years of age (Becker et al., 2004; Koch et al., 2007). Compared to the recently investigated 102 subjects the children showed, on average, clearly higher urinary phthalate metabolite levels and daily intake values. Using two different calculation models (creatinine and volume based model) children had median (maximal) intakes of 4.3 (140) and 7.8 (409) lg ⁄ kg ⁄ day for DEHP, 4.1 (76.4) and 7.6 (110) lg ⁄ kg ⁄ day for DnBP, and 0.42 (13.9) and 0.77 (31.3) lg ⁄ kg ⁄ day for BBzP, respectively (Koch et al., 2007; Wittassek et al., 2007a). Nearly one-fifth of the children showed phthalate exposure higher than the cumulative TDI for DnBP and DEHP (Fig. 2). In addition, we found that the oxidative metabolism of DEHP is dependent on age. In the children the ratios of the oxidized DEHP metabolites to MEHP generally increased with decreasing age (Becker et al., 2004). Within the 102 subjects we observed that the children at the age of 6–7 excreted on average about four times more oxidative DEHP metabolites compared to MEHP than the adults between 19 and 80 years of age (data not shown). Discussion and conclusions Our metabolism studies have shown that in the case of the long chain phthalates the oxidized metabolites are 134

the main metabolites. A great advantage of these secondary metabolites is that they are not prone to contamination. In contrast, the simple monoesters are susceptible to external contamination as they can be generated out of the omnipresent diesters. Furthermore they are only minor metabolites and have shorter halflives. For all these reasons the secondary metabolites are much more suitable biomarkers of the long chain phthalates. The proportion of the dose that is excreted via urine as the monoester and its hydroxy, oxo and carboxy products decreases with increasing alkyl chain length (Fig. 1). This may mean that a higher proportion is excreted via faeces and ⁄ or further oxidative metabolites, e.g. with two functional groups or with shortened alkyl chain may be generated (Silva et al., 2006). In the case of the short chain phthalates the simple monoesters seem to be the major metabolites. For the monoesters of DnBP and BBzP renal excretion rates of around 70% have been determined (Anderson et al., 2001). Thus, the portion of possible oxidative metabolites may be distinctly lower. For the oxidative DnBP metabolite mono(3-carboxypropyl) phthalate (3cx-MPP) we estimated a urinary excretion rate of around 0.5% of a DnBP dose administered via a pharmaceutical (Koch et al., 2004b, 2005b). The oxidative pathway of phthalate metabolism seems to be more effective in children than in adults. Neonates show a further deviation in oxidative DEHP metabolism with 5cx-MEPP being by far the predominating metabolite (Koch et al., 2006). Age-dependent metabolism of phthalates may also have relevance to health: the oxidation products are longer in the human body than the simple monoesters, they might be more toxic (Stroheker et al., 2005) and young children are a more vulnerable population group. Several biomonitoring studies have revealed the widespread exposure to several phthalates in the general population. In a recent population-based study we detected in virtually each of 102 urine samples metabolites of five phthalates. In general, the daily intakes for these phthalates were estimated to be in the range of a few micrograms per kg bodyweight and day. However, in individual cases also clearly higher values were calculated. In general, children have higher exposures to phthalates in relation to their body weight. In order to assess the exposure to DEHP and DnBP taking into consideration the additive adverse effects, we summed the percentage degrees to which the individual TDI values are reached. By the introduction of a cumulative TDI value, which is by definition equal to 100%, we may be in a better position to assess the possible health risk resulting from simultaneous exposure to several phthalates.

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M. Wittassek and J. Angerer

Phthalates: metabolism and exposure

Acknowledgements We especially thank Dr Holger Koch for his research work. We thank the Deutsche Forschungsgemeinschaft (German Research Foundation) for the financial support (DFG no. AN 107 ⁄ 16-4). We also thank the German Federal Environmental Agency who provided the urine samples from children of the 2001 ⁄ 2002 pilot study that preceded the German Environmental Survey on children (GerES IV). References AgPU (2006) Plastizicers Market Data. Arbeitsgemeinschaft PVC und Umwelt e.V., Bonn, Germany. Anderson, W., Castle, L. & Scotter, M. (2001) A biomarker approach to measuring human dietary exposure to certain phthalate diesters. Food Additives and Contaminants 18, 1068–1074. Becker, K., Seiwert, M., Angerer, J., Heger, W., Koch, H. M., Nagorka, R., Rosskamp, E., Schluter, C., Seifert, B. & Ullrich, D. (2004) DEHP metabolites in urine of children and DEHP in house dust. International Journal of Hygiene and Environmental Health 207, 409–417. Blount, B. C., Silva, M. J., Caudill, S. P., Needham, L. L., Pirkle, J. L., Sampson, E. J., Lucier, G. W., Jackson, R. J. & Brock, J. W. (2000) Levels of seven urinary phthalate metabolites in a human reference population. Environmental Health Perspectives 108, 979–982. Borch, J., Ladefoged, O., Hass, U. & Vinggaard, A. M. (2004) Steroidogenesis in fetal male rats is reduced by DEHP and DINP, but endocrine effects of DEHP are not modulated by DEHA in fetal, prepubertal and adult male rats. Reproductive Toxicology 18, 53–61. Borch, J., Axelstad, M., Vinggaard, A. M. & Dalgaard, M. (2006) Diisobutyl phthalate has comparable anti-androgenic effects to di-n-butyl phthalate in fetal rat testis. Toxicology Letters 163, 183–190. CDC (2005) Third National Report on Human Exposure to Environmental Chemicals. Department of Health and Human Services, Centers for Disease Control and Prevention (CDC), Atlanta, GA. EFSA (2005a) Opinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food (AFC) on a request from the Commission related to Di-Butylphthalate (DBP) for use in food contact materials. The EFSA Journal 242, 1–17. EFSA (2005b) Opinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food (AFC) on a request from the Commission related to Bis(2-ethylhexyl)phthalate (DEHP) for use in food contact materials. The EFSA Journal 243, 1–20. EFSA (2005c) Opinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food (AFC) on a request from the Commission related ª 2008 The Authors Journal compilation ª 2008 Blackwell Publishing Ltd



to Di-isononylphthalate (DINP) for use in food contact materials. The EFSA Journal 244, 77–83. EFSA (2005d) Opinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food (AFC) on a request from the Commission related to Butylbenzylphthalate (BBP) for use in food contact materials. The EFSA Journal 241, 1–14. EFSA (2005e) Opinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food (AFC) on a request from the Commission related to Di-isodecylphthalate (DIDP) for use in food contact materials. The EFSA Journal 241, 1–14. EFSA (2005f) Statement of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food on a request from the Commission on the possibility of allocating a group-TDI for Butylbenzylphthalate (BBP), di-Butylphthalate (DBP), Bis(2-ethylhexyl) phthalate (DEHP), di-Isononylphthalate (DINP) and di-Isodecylphthalate (DIDP). European Food Safety Authority, Parma, Italy. Gray, L. E., Jr, Ostby, J., Furr, J., Price, M., Veeramachaneni, D. N. & Parks, L. (2000) Perinatal exposure to the phthalates DEHP, BBP, and DINP, but not DEP, DMP, or DOTP, alters sexual differentiation of the male rat. Toxicological Sciences 58, 350–365. Gray, L. E., Jr, Wilson, V. S., Stoker, T., Lambright, C., Furr, J., Noriega, N., Howdeshell, K., Ankley, G. T. & Guillette, L. (2006) Adverse effects of environmental antiandrogens and androgens on reproductive development in mammals. International Journal of Andrology 29, 96–104; discussion 105– 108. Howdeshell, K. L., Furr, J., Lambright, C. R., Rider, C. V., Wilson, V. S. & Gray, L. E., Jr (2007) Cumulative effects of dibutyl phthalate and diethylhexyl phthalate on male rat reproductive tract development: altered fetal steroid hormones and genes. Toxicological Sciences 99, 190–202. Kessler, W., Numtip, W., Grote, K., Csanady, G. A., Chahoud, I. & Filser, J. G. (2004) Blood burden of di(2-ethylhexyl) phthalate and its primary metabolite mono(2-ethylhexyl) phthalate in pregnant and nonpregnant rats and marmosets. Toxicology and Applied Pharmacology 195, 142–153. Koch, H. M. & Angerer, J. (2007) Di-iso-nonylphthalate (DINP) metabolites in human urine after a single oral dose of deuterium-labelled DINP. International Journal of Hygiene and Environmental Health 210, 9–19. Koch, H. M., Rossbach, B., Drexler, H. & Angerer, J. (2003) Internal exposure of the general population to DEHP and other phthalates – determination of secondary and primary phthalate monoester metabolites in urine. Environmental Research 93, 177–185. Koch, H. M., Bolt, H. M. & Angerer, J. (2004a) Di(2-ethylhexyl)phthalate (DEHP) metabolites in human urine and serum after a single oral dose of deuterium-labelled DEHP. Archives of Toxicology 78, 123–130. Koch, H. M., Mu¨ller, J., Drexler, H. & Angerer, J. (2004b) Dibutylphthalat (DBP) in Arzneimitteln: ein bisher

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unterscha¨tztes Risiko fu¨r Schwangere und Kleinkinder. Umweltmedizin in Forschung und Praxis 10, 144–146. Koch, H. M., Bolt, H. M., Preuss, R. & Angerer, J. (2005a) New metabolites of di(2-ethylhexyl)phthalate (DEHP) in human urine and serum after single oral doses of deuterium-labelled DEHP. Archives of Toxicology 79, 367–376. Koch, H. M., Drexler, H. & Angerer, J. (2005b) DBP (Di-nbutylphthalate) in pharmaceuticals: are pregnant women and infants at risk? In: 15th Annual Conference of the International Society of Exposure Analysis, Tucson, AZ. Koch, H. M., Preuss, R. & Angerer, J. (2006) Di(2-ethylhexyl)phthalate (DEHP): human metabolism and internal exposure – an update and latest results. International Journal of Andrology 29, 155–165. Koch, H. M., Becker, K., Wittassek, M., Seiwert, M., Angerer, J. & Kolossa-Gehring, M. (2007) Di-n-butylphthalate and butylbenzylphthalate – urinary metabolite levels and estimated daily intakes: pilot study for the German Environmental Survey on children. Journal of Exposure Science and Environmental Epidemiology 17, 378–387. Lee, K. Y., Shibutani, M., Takagi, H., Kato, N., Takigami, S., Uneyama, C. & Hirose, M. (2004) Diverse developmental toxicity of di-n-butyl phthalate in both sexes of rat offspring after maternal exposure during the period from late gestation through lactation. Toxicology 203, 221–238. Saillenfait, A. M., Sabate, J. P. & Gallissot, F. (2006) Developmental toxic effects of diisobutyl phthalate, the methylbranched analogue of di-n-butyl phthalate, administered by gavage to rats. Toxicology Letters 165, 39–46. Schmid, P. & Schlatter, C. (1985) Excretion and metabolism of di(2-ethylhexyl)phthalate in man. Xenobiotica 15, 251–256. Sharpe, R. M. & Irvine, D. S. (2004) How strong is the evidence of a link between environmental chemicals and adverse effects on human reproductive health? British Medical Journal 328, 447–451. Silva, M. J., Barr, D. B., Reidy, J. A., Malek, N. A., Hodge, C. C., Caudill, S. P., Brock, J. W., Needham, L. L. & Calafat, A. M. (2004) Urinary levels of seven phthalate metabolites in the U.S. population from the National Health and Nutrition Examination Survey (NHANES) 1999–2000. Environmental Health Perspectives 112, 331–338. Silva, M. J., Kato, K., Wolf, C., Samandar, E., Silva, S. S., Gray, E. L., Needham, L. L. & Calafat, A. M. (2006) Urinary biomarkers of di-isononyl phthalate in rats. Toxicology 223, 101–112. Stroheker, T., Cabaton, N., Nourdin, G., Regnier, J. F., Lhuguenot, J. C. & Chagnon, M. C. (2005) Evaluation of antiandrogenic activity of di-(2-ethylhexyl)phthalate. Toxicology 208, 115–121. Wittassek, M., Heger, W., Koch, H. M., Becker, K., Angerer, J. & Kolossa-Gehring, M. (2007a) Daily intake of di(2-ethylhexyl)phthalate (DEHP) by German children – a comparison of two estimation models based on urinary DEHP metabolite levels. International Journal of Hygiene and Environmental Health 210, 35–42.

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Wittassek, M., Wiesmuller, G. A., Koch, H. M., Eckard, R., Dobler, L., Muller, J., Angerer, J. & Schluter, C. (2007b) Internal phthalate exposure over the last two decades – a retrospective human biomonitoring study. International Journal of Hygiene and Environmental Health 210, 319– 333. Wolfe, G. W. & Layton, K. A. (2003) Multigeneration Reproduction Toxicity Study in Rats: Multigenerational Reproductive Assessment by Continuous Breeding when Administered to Sprague–Dawley Rats in the Diet. TherImmune Research Corporation, Gaithersburg, MD.

Panel discussion D. Waxman What is known about the intrinsic toxicities of the oxidized phthalates compared to the parent monoesters? J. Angerer The relative toxicities are not known and must be studied, however, reports suggest that the oxidative products are the most toxic. D. Waxman Are the oxidative products formed in the liver, for example via hepatic cytochrome P450 metabolism? J. Angerer This is probably the case but it has not been investigated directly. N.E. Skakkebæk Why are children more exposed to phthalates? Perhaps they consume more than adults. J. Angerer Children have high levels but we do not know why. Diet accounts for 99.5% of DHP, and children’s diet may be different from adults. They may also use different personal care products. E. Gray Are phthalates present in baby products? J. Angerer Yes

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ª 2008 The Authors International Journal of Andrology 31, 131–138

M. Wittassek and J. Angerer

Phthalates: metabolism and exposure

S. Swan

A.M. Calafat

We asked mothers about their use of baby care products. The number of personal care products used by the mother on the baby was directly and significantly related to the urinary metabolite concentration of several phthalates, in particular MEP.

What is the best way to assess exposure to the isomeric phthalates DINP and DIDP, which appears to be increasing? There are many different isomeric metabolites but only a limited number of standard compounds are available.

A.A. Jensen

J. Angerer

We have made a report to the Danish Environmental Protection Agency about the indoor environment. Children are more exposed to phthalates because of toys, consumer products and building materials.

DINP and DIDP exposure should be measured in the same way although lack of standard material is a problem. However, uptake of these substances is much less, therefore their environmental effect is less important.

J. Angerer

J. Ko¨hrle

Phthalates in air are a minor component in exposure and absorption. In our German study there was no correlation between dust content and excretion of phthalate metabolites.

Is there any difference in the protein binding of absorbed phthalates and their metabolic products? If there is protein binding of a large fraction of these chemicals there will be different accessibility to different organs such as the brain because of the blood brain barrier compared to the free form.

A.-M. Andersson Children have a relatively larger surface area for absorbing phthalates. What is the best biomarker of phthalate exposure? Russ Hauser suggested that the ratio of different metabolites in the urine is the best measure of exposure to harmful products, therefore, more than 1 marker and the ratio should be measured.

J. Angerer We have no experience of serum content of phthalates and their binding to protein. In our urine sample we perform a hydrolysis step so that the total amount of excreted phthalate is measured but we do not know how much is protein bound.

J. Angerer Results are much more accurate if 2 or 3 metabolites are measured in urine to assess exposure to DEHP: there is no better parameter. Maternal urine levels are the best measure of fetal exposure.

R. Sharpe You indicated that following equivalent levels of exposure, the internal concentration of phthalates in humans is 100 times greater than the concentration in rats, and greater than the concentration in marmosets.

A.M. Calafat Not all metabolites need to be analysed in the urine to assess phthalate exposure. For example, the hydroxyland oxo-metabolites of DEHP are produced by a similar pathway and their levels are highly correlated. The carboxy-metabolites are produced through a different pathway involving omega oxidation. Measuring the carboxy-metabolite and only one of the hydroxyl- or oxo-metabolites will reduce the work load and still give accurate results. J. Angerer I agree. ª 2008 The Authors Journal compilation ª 2008 Blackwell Publishing Ltd



J. Angerer Rats and marmosets were exposed to much higher levels than humans, but if we correct for the dose of exposure, the relative internal concentration is much higher in humans. R. Sharpe This must be considered when calculating safety factors. The safe level determined in rodents and marmosets must be reduced by a factor of 100 when assessing the safe level in humans because of differential absorption ratios.

International Journal of Andrology 31, 131–138

137

Phthalates: metabolism and exposure

M. Wittassek and J. Angerer

E. Gray

H. Leffers

When calculating safe exposure levels (TDI and RfD) from animal experiments, there is a built in uncertainty factor of 10X to account for species difference. It now appears that science on phthalates presented in this talk has shown that this factor must be adjusted and should be raised to 100X. Following dermal application and absorption of phthalates, the metabolites of the parent material were measured. Oral phthalates are metabolized by non specific esterases in the gut then by liver enzymes before entering the systemic circulation. Are dermally applied phthalates metabolized in the skin or by blood lipases, and how much exposure to the parent compound occurs?

Phthalates and their known metabolites are charged particles and I cannot understand how they can pass through the cell membrane to cause damage within the cell. Are there any non charged polar metabolites which might be responsible for the toxicity?

P. Foster

E. Gray

There are non specific esterases in red blood cells. The phthalate monoesters are metabolized very rapidly.

Some pharmacokinetic studies have assessed charge and distribution in the body. I do not know how a charged particle enters the cell. In vitro studies have demonstrated intracellular effects, therefore, some toxic compounds enter the cell.

E. Gray Is there any difference in excretion profiles following oral and intravenous application of phthalates?

J. Angerer There is a different profile of phthalates and their metabolites in urine, serum and amniotic fluid, and I am not sure which are the toxic metabolites and which enter the cell. The analysis of metabolites in urine is a measure of exposure without indicating possible toxicity.

H. Leffers We have examined the uptake of these compounds, but following addition of large quantities of monophthalates to culture media we have never identified any phthalate within cells.

J. Angerer There is no difference. G. Lyons In relation to the higher levels of phthalates in children, has there been any recent monitoring of the levels in sweets? Previously it was found that elevated levels occurred arising from sweet and chocolate wrappings.

A.A. Jensen There has been speculation that phthalates have reproductive toxicity because of the chemical similarity to thalidomide.

J. Angerer

E. Gray

We are aware that confectionary wrappings may contain phthalates but we are unaware of which products are affected and which phthalates are involved. We do not know of any recent studies.

Thalidomide is inactive in the rodent as a teratogen although some effects are seen in rabbits. This suggests that the effects of phthalates in rats are not due to thalidomide-like activity.

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Journal compilation ª 2008 Blackwell Publishing Ltd



ª 2008 The Authors International Journal of Andrology 31, 131–138