Human biomonitoring of phthalate exposure in Austrian children and adults and cumulative risk assessment

International Journal of Hygiene and Environmental Health 218 (2015) 489–499

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Human biomonitoring of phthalate exposure in Austrian children and adults and cumulative risk assessment Christina Hartmann a,b,∗ , Maria Uhl a , Stefan Weiss a , Holger M. Koch c , Sigrid Scharf a , Jürgen König b a

Environment Agency Austria, Spittelauer Lände 5, 1090 Vienna, Austria Department of Nutritional Sciences, University of Vienna, Althanstraße 14, 1090 Vienna, Austria c Institute for Prevention and Occupational Medicine of the German Social Accident Insurance, Institute of the Ruhr-University Bochum (IPA), Bürkle-de-la-Camp-Platz 1, 44789 Bochum, Germany b

a r t i c l e

i n f o

Article history: Received 3 October 2014 Received in revised form 14 April 2015 Accepted 14 April 2015 Keywords: Urinary phthalate metabolites Human biomonitoring Estimated daily intake Cumulative risk assessment HPLC–MS/MS

a b s t r a c t Phthalates are a class of chemicals widely used as plasticisers in a multitude of common consumer products. Through contact with such products, people are regularly exposed to phthalates, which are suspected to contribute to adverse health effects, particularly in the reproductive system. In the present study, 14 urinary phthalate metabolites of 10 parent phthalates were analysed by HPLC–MS/MS among the Austrian population aged 6–15 and 18–81 years in order to assess phthalate exposure. In the total study population, ranges of urinary phthalate metabolite concentrations were n.d.–2,105 ␮g/l (median 25 ␮g/l) for monoethyl phthalate (MEP), n.d.–88 ␮g/l (10 ␮g/l) for monon-butyl phthalate (MnBP), n.d.–248 ␮g/l (28 ␮g/l) for mono-isobutyl phthalate (MiBP), n.d.–57 ␮g/l (1.8 ␮g/l) for mono-benzyl phthalate (MBzP), n.d.–20 ␮g/l (n.d.) for mono-(2-ethylhexyl) phthalate (MEHP), n.d.–80 ␮g/l (2.6 ␮g/l) for mono-(2-ethyl-5-hydroxyhexyl) phthalate (5OH-MEHP), n.d.–57 ␮g/l (1.9 ␮g/l) for mono-(2-ethyl-5-oxohexyl) phthalate (5oxo-MEHP), n.d.–219 ␮g/l (11 ␮g/l) for mono(5-carboxy-2-ethylpentyl) phthalate (5cx-MEPP), n.d.–188 ␮g/l (1.6 ␮g/l) for 3-carboxy-mono-proply phthalate (3cx-MPP), n.d.–5.5 ␮g/l (n.d.) for mono-cyclohexyl phthalate (MCHP), n.d.–4.5 ␮g/l (n.d.) for mono-n-pentyl phthalate (MnPeP), n.d.–3.4 ␮g/l (n.d.) for mono-n-octyl phthalate (MnOP), n.d.–13 ␮g/l (n.d.) for mono-isononyl phthalate (MiNP), and n.d.–1.1 ␮g/l (n.d.) for mono-isodecyl phthalate (MiDP). Generally, children exhibited higher levels of exposure to the majority of investigated phthalates, except to MEP, which was found in higher concentrations in adults and senior citizens at a maximum concentration of 2,105 ␮g/l. Individual daily intakes were estimated based on urinary creatinine and urinary volume excretion and were then compared to acceptable exposure levels, leading to the identification of exceedances of mainly the Tolerable Daily Intakes (TDI), especially among children. The execution of a cumulative risk assessment based on Hazard Indices showed cause for concern mainly for children, as well as in rare cases for adults. Although phthalate exposure seems to have decreased in previous years, the wide distribution and existing exceedances of acceptable levels indicate that phthalate exposure should be further monitored in order to identify exposure sources and enable appropriate minimisation measures. © 2015 Elsevier GmbH. All rights reserved.

Introduction Phthalates are the di-esters of 1,2-benzenedicarboxylic acid and a group of man-made environmental chemicals which are produced worldwide in high annual amounts and are primarily used as plasticisers in the production of polyvinyl chloride

∗ Corresponding author at: Environment Agency Austria, Spittelauer Lände 5, 1090 Vienna, Austria. Tel.: +43 1 31304 3606. E-mail address: [email protected] (C. Hartmann). http://dx.doi.org/10.1016/j.ijheh.2015.04.002 1438-4639/© 2015 Elsevier GmbH. All rights reserved.

(PVC), accounting for 87% of the annual global plasticiser production (EAG (Environmental Agency Germany), 2011). Depending on their molecular weight, they are used for divergent applications and can be found in a wide range of consumer products (ECB (European Chemicals Bureau), 2003, 2008; ECHA (European Chemicals Agency), 2009, 2013c; Danish EPA (Danish Environmental Protection Agency), 2011; Hauser and Calafat, 2005; NRC (National Research Council), 2008). Because phthalates are not chemically bound to the polymer structures of products and articles, they are able to migrate continuously from surfaces into food and environment (Navarro et al.,

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2010; Wormuth et al., 2006), which can lead to human exposure by various routes. Following uptake into the body, phthalates are rapidly absorbed, metabolised by hydrolysis and subsequent oxidation and excreted via urine and faeces. More than 95% of an administrated oral dose of a phthalate is eliminated as the corresponding metabolite(s) through renal excretion within 24 h (Hauser and Calafat, 2005; Koch and Angerer, 2012; Zeman et al., 2013). Although they are rapidly excreted, particular attention has been paid to phthalates for many years especially because of their endocrine-disrupting effects and toxicity to reproduction (EC (European Commission), 2011; Danish EPA (Danish Environmental Protection Agency), 2013; Kortenkamp et al., 2011). Several phthalates have been identified as endocrine-disrupting chemicals (EDCs) acting as anti-androgens, estrogens, anti-estrogens or inhibitors of steroidogenic enzymes in the body, as well as with thyroid hormones and their related receptors (Fisher, 2004). Antiandrogenic and/or thyroid endocrine disrupting phthalates include DnBP, DiBP, BBzP and DEHP, and additionally, DEP is included in the EU list of potential endocrine disruptors (Danish EPA (Danish Environmental Protection Agency), 2013). Studies in experimental animals as well as in humans have shown phthalates leading to diverse adverse health effects reported elsewhere (ECB (European Chemicals Agency), 2008; ECHA (European Chemicals Agency), 2008, 2013a, 2013b, 2014a, 2014b; Braun et al., 2013; Bornehag and Nanberg, 2010; Hauser and Calafat, 2005; Jaakkola and Knight, 2008; Jurewicz and Hanke, 2011; Kay et al., 2013; Kimber and Dearman, 2010; Latini et al., 2006; Matsumoto et al., 2008; Meeker et al., 2009; Swan, 2008). Some phthalates such as DEHP and DiBP are known to induce peroxisome proliferator-activated receptors (PPAR), which play important roles in the regulation of a variety of biological processes, such as adipocyte proliferation and differentiation, glucose homeostasis, intracellular trafficking of lipids and their metabolism, inflammatory responses, vascular functions and embryonic and fetal development (Lau et al., 2010). Because of the concern regarding phthalates as substances produced and used in high amounts and in a wide range of consumer products, as well as their identified adverse effects on human health, investigations of exposures to populations are of high importance. The present study is one of the first comprehensive investigations of the phthalate exposure of the population in Austria based on data and samples from the Austrian Study of Nutritional Status 2010/2012 (ASNS) performed by the Department of Nutritional Sciences of the University of Vienna. Therefore, phthalate metabolite concentrations were analysed in spontaneous urine samples of a large sample including children and adolescents (6–15 years), adults (18–64 years) and senior citizens (≥65 years) and were used for calculations of daily intakes (DIs) and cumulative risk assessment estimations. The observed findings were compared to Tolerable Daily Intake values (TDI) set out by the European Food Safety Authority (EFSA) (EC (European Commission), 2013; EFSA (European Food Safety Authority), 2005a, 2005b, 2005c, 2005d, 2005e), to the Reference Doses (RfD) set out by the U.S. EPA (United States Environmental Protection Agency) (2007b) and to the References Doses for Anti-Androgenicity (RfD AA) established by Kortenkamp and Faust (2010). Additionally, comparisons with results obtained from several studies, especially from other European countries, were performed in order to identify potential differences and trends in phthalate exposure. Materials and methods Study design and study population The Austrian Study on Nutritional Status (ASNS) performed by the Department of Nutritional Sciences of the University of

Vienna comprised a total of 1002 participants including 387 male and female children and adolescents aged 6–15 years, 419 male and female adults aged 18–64 years and 196 male and female senior citizens aged 65–81 years through a quota sampling of a cross-sectional study. The recruitment of children and adolescents occurred in selected Austrian schools in almost all federal states and of adults and senior citizens via companies, municipal offices, clubs and retirement homes. Detailed recruitment and sampling procedures were described in Elmadfa et al. (2012). The fieldwork took place between 2010 and 2012, with data on nutrition, education, employment and health status being collected via questionnaires, and spontaneous urine samples taken were collected before midday. For the analysis of phthalate exposure, the surveyed data and the urine samples of more than a half of the within ASNS recruited participants were sent to the Environment Agency Austria (EAA) in dependence of the availability of sufficient sample material, including a total of 595 participants comprising 251 children and adolescents aged 6–15 years (142 males and 109 females), 272 adults aged 18–64 years (mean age 39.1 years; 110 males and 162 females), and 72 senior citizens aged 65–81 years (mean age 71.4 years; 34 males and 38 females) being investigated. Due to the recruitment procedure, the group of children and adolescents was further divided into two subgroups according to the level of education: Children I for those aged 6–8 years from the 1st and 2nd levels of education, and Children II for those aged 7–15 years from the 3rd to 8th levels of education. For this reason, small overlaps in age exist between members of the two subgroups. Questionnaires completed by younger children were amended by additional questionnaires given to their respective parents. Study participants originated from almost all Austrian federal states. For investigations of potential regional differences, the study population was grouped according to their residence, with 229 participants living in rural and 284 participants in urban or suburban areas. The study was approved by the ethics commission of the City of Vienna (EK 10 037 0310).

Chemical analysis Concentrations of 14 urinary phthalate metabolites (Table 1) were measured by high-performance liquid chromatography tandem-mass spectrometry (HPLC–MS/MS) for simultaneous determination of several metabolites after enzymatic hydrolysis with beta-glucuronidase, an implemented and accredited method developed by the EAA, which was adapted from Koch et al. (2003c) and Preuss et al. (2005), and was extended to the metabolites MCHP, MnPeP, MiNP, MiDP and 3cx-MPP. Validation data for the metabolites also described in Koch et al. (2003c) and Preuss et al. (2005) are comparable. Detailed description of analysis and validation data is published in Hartmann (2014). The HPLC system used in this study was an Agilent Technologies 1290 Infinity Series (Agilent Technologies, Santa Clara, CA, USA), and the MS detector system was an AB Applied Biosystem MDS SCIEX 4000 QTRAP LC/MS/MS System (AB Sciex Technologies, Framingham, MA, USA) which allowed detection through specific mass transitions in electrospray (ESI) negative mode, and quantification in multiple reaction monitoring (MRM) mode. The analytical column was a Kinetex 2.6␮ Phenyl-Hexyl 100A LC Column (Phenomenex, USA). The limit of quantitation (LOQ) for each substance was determined according to DIN 32645 (see DIN, 2008). Limits of detection (LOD) were set as half of the respective LOQ and were 2.5 ␮g/l for MEP, 0.53 ␮g/l for MnBP, 0.59 ␮g/l for MiBP and MBzP, 0.69 ␮g/l for MEHP, 0.79 ␮g/l for 5OH-MEHP, 0.56 ␮g/l for 5oxo-MEHP, 0.46 ␮g/l for 5cx-MEPP, 1.6 ␮g/l for 3cx-MPP, 0.51 ␮g/l for MCHP, 0.55 ␮g/l

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Table 1 Investigated phthalate metabolites. Parent phthalate

Primary metabolite

Diethyl phthalate (DEP) Di-(2-ethylhexyl) phthalate (DEHP)

Monoethyl phthalate (MEP) Mono-(2-ethylhexyl) phthalate (MEHP)

Di-n-butyl phthalate (DnBP) Di-isobutyl phthalate (DiBP) Butyl benzyl phthalate (BBzP) Di-cyclohexyl phthalate (DCHP) Di-n-pentyl phthalate (DnPeP) Di-n-ocytl phthalate (DnOP) Di-isononyl phthalate (DiNP) Di-isodecyl phthalate (DiDP) DnBP, DnOP, and/or DiNPa

Mono-n-butyl phthalate (MnBP) Mono-isobutyl phthalate (MiBP) Mono-benzyl phthalate (MBzP) Mono-cyclohexyl phthalate (MCHP) Mono-n-pentyl phthalate (MnPeP) Mono-n-octyl phthalate (MnOP) Mono-isononyl phthalate (MiNP) Mono-isodecyl phthalate (MiDP)

a

Secondary metabolite Mono-(2-ethyl-5-hydroxyhexyl) phthalate (5OH-MEHP) Mono-(2-ethyl-5-oxohexyl) phthalate (5oxo-MEHP) Mono-(5-carboxy-2-ethylpentyl) phthalate (5cx-MEPP)

3-carboxyl-mono-propyl phthalate (3cx-MPP)

3cx-MPP is a metabolite of three phthalates (DnBP, DnOP and DiNP).

for MnPeP, 0.45 ␮g/l for MnOP and 0.47 ␮g/l for MiNP and MiDP, respectively. The analytical method has been proven successfully by several inter-laboratory comparison tests within the European COPHES/DEMOCOPHES projects (Consortium to Perform Human Biomonitoring on a European Scale/Demonstration Of A Study To Coordinate And Perform Human Biomonitoring On An European Scale), see Schindler et al. (2014). The between-day imprecision and accuracy were reasonable, with the relative standard deviations (RSD) ranging between 10.8 and 19.8% depending on the specific substance, and the mean relative recoveries ranging between 94.6 and 115.8%. Individual creatinine levels in urine samples were measured via a clinical diagnosis system (Vitros, Ortho-Clinical Diagnostics, Germany) using a colorimetric assay and were provided by the Department of Nutritional Sciences. Statistical analysis Statistical analysis was performed using IBM® SPSS® Statistics Version 21. Descriptive statistics (ranges, medians, 95th percentiles) were determined for phthalate metabolite concentrations expressed in ␮g/l urine, as well as for creatinine-adjusted phthalate metabolite concentrations expressed in ␮g/g creatinine. For data treatment, concentrations
provided from Geigy (1983) for children and adolescents, and from Hays et al. (2011) for adults and senior citizens, and reference values for 24-h urinary creatinine excretion were provided from Remer et al. (2002) for children and adolescents, and from Koch et al. (2003a) for adults and senior citizens. The used molar fraction values (FUE ) were derived from studies investigating urinary phthalate metabolite concentrations 24 or 48 h after oral application of known doses of respective labelled parent compounds in volunteers and were 0.69 for MEP (Koch and Angerer, 2012), 0.73 for MBzP (Anderson et al., 2001), 0.84 for MnBP, 0.7 for MiBP (Koch et al., 2012) and 0.062, 0.149 and 0.109 for the DEHP metabolites MEHP, 5OH-MEHP and 5oxo-MEHP, respectively (Anderson et al., 2011). Daily intakes were estimated for DEP, BBzP, DnBP, DiBP and DEHP and were compared with existing acceptable exposure levels for phthalates that can be ingested daily over the course of a lifetime without resulting in significant risk to human health: the Tolerable Daily Intake (TDI) set out by EFSA, the Reference Dose (RfD) and the Reference Dose for Anti-Androgenicity (RfD AA) established by Kortenkamp and Faust (2010). Individual acceptable exposure levels are listed in Table 5. Because EFSA does not currently offer a TDI for DiBP, the TDI of DiBP is set out in analogy to DnBP (Koch et al., 2011).

Cumulative risk assessment estimates For accounting effects of combined exposures, the dose-addition concept by using the Hazard Index (HI) for the estimation of the overall potential for non-carcinogenic effects of several phthalates was used (Kortenkamp and Faust, 2010; U.S. EPA (United States Environmental Protection Agency), 1989). The HI is defined as the ratio of the sum of Hazard Quotients (HQ) to the acceptable level (AL) of exposure of a substance. For cumulative risk assessment calculation, the formula published in Kortenkamp and Faust (2010) was used HI =

n  EL

i

i=1

ALi

(3)

where EL is the exposure level (or daily intake) of a substance, AL is the acceptable level of exposure (TDI or RfD AA), and n is the number of substances in the mixture. When HI exceeds the value of 1, the total dose of all substances in the mixture exceeds the considered acceptable exposure level (Kortenkamp and Faust, 2010).

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98.6 97.3 100 82.2 37.0 98.6 93.2 100 86.3 8.2 4.1 8.2 16.4 23.3 n, Number of analysed samples; n.d., not detectable; LOQ, quantitation limit; P95, 95th percentile, DR, detection rate [%].

P95

1,188 57 111 13 6.6 31 20 81 16
Median Range

n.d.–2,105 n.d.–85 2.5–152 n.d.–17 n.d.–12 n.d.–76 n.d.–55 1.0–134 n.d.–38 n.d.–
DR P95 Median

16 27 54 5.3
Senior citizens (n = 72)

DR P95 Median Range

Adults (n = 272)

DR P95 Range Range

Median

Children II (n = 220) Children I (n = 31) Metabolite

Table 2 Concentrations of several phthalate metabolites in spontaneous urine samples of the Austrian population (ranges, medians, 95th percentiles in ␮g/l; detection rate in % positive detected samples).

The results of the measurements of 14 phthalate metabolites (representing exposure to 10 parent phthalates) in altogether 595 urine samples of children and adolescents, as well as of adults and senior citizens are illustrated in Table 2, in ␮g/l. Creatinine-adjusted phthalate metabolite concentrations are shown in Table 3, in ␮g/g. MEP, MnBP, MiBP, MBzP, MEHP, 5OH-MEHP, 5oxo-MEHP, 5cxMEPP and 3cx-MPP were detected in the majority of the samples, especially among children and adolescents, whereas detection rates for MCHP, MnOP, MnPeP, MiNP and MiDP were considerably lower (Table 2). These findings are in line with previous determinations of phthalate metabolites in other populations in Europe and other parts of the world, e.g. summarised in Koch and Calafat (2009) and Wittassek et al. (2011). In general, our results matched most with findings from Denmark in the same sampling period (2011) (see Frederiksen et al., 2013) and support the observation of decreasing trends in recent years also shown in other study populations (e.g. CDC (Center of Disease Control Prevention), 2014; Frederiksen et al., 2013). Investigations in German children in the same sampling period showed partially higher results compared to children from the Children I group (Fromme et al., 2013b). Urinary levels of MiBP exceeding those of its isomer MnBP have been observed only in recent years and are possibly related to the substitution of DnBP with DiBP (Kasper-Sonnenberg et al., 2014). The low detection rates of the monoester metabolites of DiNP and DiDP can be explained by earlier findings indicating that these metabolites are only minor metabolites of the respective high molecular weight phthalates excreted in urine. Oxidized metabolites are in the meantime known to be the preferred metabolites excreted in urine (Koch et al., 2007; Leng et al., 2014) and a broad excretion of these metabolites in urine samples from the general population has been reported previously. Consequently, these metabolites will be included in future measurements. As shown in Table 4, which depicts correlation coefficients between several selected urinary phthalate metabolites, statistically significant correlations (Spearman correlation, p = 0.01) were identified for most of the metabolites, except for MnOP, MiNP and MiDP (not shown in Table 4), where either no or only few correlations were observed. Thus, participants were not solely exposed to single compounds, but in general exhibited a high degree of exposure to the majority of the investigated phthalate metabolites. The metabolites of DEHP correlated consequentially among each other, with the strongest correlations being shown between the secondary ones. The correlation between the metabolite MnBP and its isomer MiBP was also strong. A possible explanation for this finding is the interchangeable use of DnBP and DiBP, and therefore similar sources and routes of exposure can be expected (Lorber and Koch, 2013). Additionally, correlations between MnBP, MiBP and MBzP and the metabolites of DEHP suggested a similar origin of exposure. Contrarily, no or only weak correlations were shown for MEP, which is an indication for the use of personal care products and cosmetics as the major source for its parent compound. Between the four different study population groups of Children I (mean age 7 years, n = 31), Children II (mean age 11 years, n = 220), Adults (mean age 39 years, n = 272) and Senior citizens (mean age 71 years, n = 72) we observed several statistically significant differences (p = 0.000 performing Kruskal–Wallis tests) in urinary metabolite levels. Generally, urinary phthalate metabolite levels decreased with increasing age for all metabolites, except for MEP (Table 2). This general age-related trend of lower phthalate exposure levels in adults seemed to stop or even reverse with the senior citizens. Our findings warrant further investigations in this field, especially because published data on the phthalate exposure

n.d.–447 2.0–70 5.6–177 n.d.–53 n.d.–11
Urinary phthalate metabolite levels

MEP MnBP MiBP MBzP MEHP 5OH-MEHP 5oxo-MEHP 5cx-MEPP 3cx-MPP MCHP MnPeP MnOP MiNP MiDP

DR

Results and discussion

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493

Table 3 Creatinine-adjusted concentrations of several phthalate metabolites in spontaneous urine samples of the Austrian population (ranges, medians, 95th percentiles in ␮g/g creatinine; detection rate in % positive detected samples). Metabolite

MEP MnBP MiBP MBzP MEHP 5OH-MEHP 5oxo-MEHP 5cx-MEPP 3cx-MPP MCHP MnPeP MnOP MiNP MiDP

Children I (n = 30)

Children II (n = 215)

Adults (n = 269)

Senior citizens (n = 69)

Range

Median

P95

Range

Median

P95

Range

Median

P95

Range

Median

P95

n.d.–336 6.1–516 18–494 n.d.–67 n.d.–17 2.5–99 1.8–62 7.1–135
20 33 70 7.0 1.3 18 13 31 5.2 n.d. n.d. n.d. n.d. n.d.

331 371 461 64 15 86 60 134 31
n.d.–1,645 n.d.–699 n.d.–1,086 n.d.–53 n.d.–23 n.d.–131 n.d.–94 2.7–179 n.d.–206 n.d.–6.9 n.d.–3.4 n.d.–3.6 n.d.–5.7 n.d.–2.2

21 12 31 2.5 0.94 31 3.3 15
114 73 213 26 8.5 213 25 67 17 1.2
n.d.–1,276 n.d.–78 n.d.–497 n.d.–40 n.d.–6.1 n.d.–28.6 n.d.–18 n.d.–131 n.d.–122 n.d.–5.2 n.d.–2.2 n.d.–1.3 n.d.–45 n.d.–3.1

29 6.5 21 1.2 n.d. 1.6
335 35 104 9.8 3.4 9.6 6.3 24 18 1.3 n.d.
n.d.–3,394 n.d.–98 4.6–241 n.d.–14 n.d.–11 n.d.–72 n.d.–52 1.8–150 n.d.–36 n.d.–
32 12 27 1.4 n.d. 2.9 1.6 9.2
1,012 54 152 12 6.6 31 17 71 21
n, Number of analysed samples; n.d., not detectable; LOQ, quantitation limit; P95, 95th percentile.

Table 4 Correlations between urinary phthalate metabolite concentrations (␮g/l) among the total study population (Spearman rank coefficients) (n = 595)..

MEP MnBP MiBP MBzP MEHP 5OH-MEHP 5oxo-MEHP 5cx-MEPP **

MnBP

MiBP

MBzP

MEHP

5OH-MEHP

5oxo-MEHP

5cx-MEPP

3cx-MPP

0.120**

0.130** 0.715**

0.034 0.628** 0.444**

−0.015 0.471** 0.464** 0.442**

0.067 0.627** 0.461** 0.626** 0.566**

0.030 0.626** 0.477** 0.637** 0.589** 0.939**

0.129** 0.548** 0.420** 0.569** 0.527** 0.814** 0.830**

0.073 0.374** 0.240** 0.269** 0.176** 0.344** 0.331** 0.384**

Correlation is significant at 0.01 level.

of senior citizens is sparse. However, these results are similar to findings from other studies. In general, children are exposed to phthalates to a greater degree than adults (Wittassek and Angerer, 2008). In the National Health and Nutrition Examination Survey (NHANES) 1999–2000, significantly higher phthalate metabolite levels (MnBP, MBzP and MEHP) were reported in the urine of children than in that of adults, but lower MEP levels, and also higher levels of several phthalates in females compared to males (Silva et al., 2004). Several consumer products such as toys, school supplies, plastic gloves, or paints, as well as food are important sources of exposure to various phthalates, which lead to more frequent contact for children (Wormuth et al., 2006). In addition, house dust might be a potential source of phthalate exposure (Bornehag et al., 2004; Kolarik et al., 2008; Wormuth et al., 2006). Additionally, physiology, developmental stages and behaviour of children may contribute to higher exposure levels and be reasonable factors (NRC (National Research Council), 2008). An explanation for higher urinary MEP levels in adults is the more likely use of cosmetics, personal care products and other MEP-containing consumer products (Heudorf et al., 2007). Regarding sex, statistically significant differences were observed for both creatinine-adjusted and creatinine-unadjusted MEP (p = 0.045 each) and creatinine-unadjusted MnBP (p = 0.045) in the group of Children I (15 females, 16 males) and for creatinineunadjusted MBzP (p = 0.021), MnBP (p = 0.000), MiBP (p = 0.045), 5oxo-MEHP (p = 0.008) and 5OH-MEHP in adults (162 females, 110 males) (Mann–Whitney U test) with females exhibiting higher internal exposures than males. For the purpose of determining possible differences resulting from area of residence, the study population was divided into two groups comprising participants from (sub)urban (n = 284) and rural areas (n = 229), respectively. Among the total study population, significant differences were found for unadjusted and

creatinine-adjusted MEP (p = 0.000 and 0.042, respectively), and the creatinine-adjusted DEHP metabolites 5oxo-MEHP (p = 0.006) and 5OH-MEHP (p = 0.021), as well as for the unadjusted metabolite 5cx-MEPP (p = 0.036) (Mann–Whitney U tests). For all named phthalate metabolites, participants from (sub)urban areas exhibited significantly higher exposure levels than participants from rural areas, which could support the hypothesis of higher environmental phthalate levels in areas with higher population density (Staples, 2003), and indicate the possibility of significant demographic variations of exposure. Daily intake estimates The estimation of individual daily phthalate intakes among the study population was performed according to two different calculation models derived from Koch et al. (2007) for creatinine excretion and urinary volume excretion, respectively. Ranges, medians and 95th percentiles (P95) for children, adolescents, adults and senior citizens for the creatinine-based model and for the volume-based model are given in Table 5. Daily intakes were compared with different acceptable exposure levels (TDI, RfD and RfD AA) in order to elevate potential exceedances. Concerning age, daily intake values decreased with increasing age for most investigated phthalates (DnBP, DiBP, BBzP and DEHP) based on both calculation models. Median daily intakes were about two to four times higher in the Children I group compared to the adults and senior citizens, and P95 values were about two to five times higher. Only for DEP were intakes higher in adults compared to the children by a factor of two to four. Based on the creatinine-based calculation model, phthalates with the highest daily intake values were DEHP in Children I (median 3.3 ␮g/kg bw/d; P95 15 ␮g/kg bw/d) and Children II (median 1.3 ␮g/kg bw/d; P95 7.2 ␮g/kg bw/d), but DEP in adults

DEHP

a

BBzP

DiBP

DnBP

Relative cumulative frequency (%)

bw: Bodyweight; EFSA: European Food Safety Authority; n: number of samples; RfD: Reference Dose; RfD AA: Reference Dose for Anti-Androgenicity; TDI: Tolerable Daily Intake; U.S. EPA: United States Environmental Protection Agency; P95: 95th percentile. a Based on urinary concentrations of MEHP, 5OH-MEHP, 5oxo-MEHP and 5cx-MEPP. b Sources: EC (European Commission) (2013); EFSA (European Food Safety Authority) (2005a, 2005b, 2005c, 2005d, 2005e). c Sources: U.S. HHS (U.S. Department of Health and Human Services) (2001), U.S. EPA (United States Environmental Protection Agency) (2007a, 2007b). d Sources: Kortenkamp and Faust (2010), Søeborg et al. (2012).

30 20 50

330 200 500

200 – –

100 100 10

– DEP

RfD (U.S. EPA)c

800

Creatinine-based Volume-based Creatinine-based Volume-based Creatinine-based Volume-based Creatinine-based Volume-based Creatinine-based Volume-based

0.0–10 (0.53; 9.9) 0.0–19 (0.65; 13) 0.15–15 (0.84; 10) 0.07–2.6 (0.99; 2.4) 0.52–17 (2.3; 14) 0.25–7.9 (2.4; 6.9) 0.0–2.1 (0.21; 1.9) 0.0–2.2 (0.22; 1.9) 0.54–16 (3.3; 15) 0.26–9.1 (3.3; 7.7)

0.0–8.9 (0.61; 3.1) 0.0–15 (0.70; 3.5) 0.0–19 (0.34; 1.8) 0.0–2.5 (0.40; 1.6) 0.0–34 (1.1; 7.0) 0.0–7.1 (1.3; 4.7) 0.0–1.3 (0.08; 0.77) 0.0–1.8 (0.09; 0.78) 0.13–21 (1.3; 7.2) 0.19–16 (1.5; 6.9)

0.0–49 (0.9; 11) 0.0–42 (1.3; 14) 0.0–2.0 (0.24; 1.1) 0.0–2.4 (0.28; 1.4) 0.0–16 (0.78; 3.4) 0.0–12 (0.99; 4.6) 0.0–1.0 (0.04; 0.23) 0.0–12 (0.99; 4.6) 0.0–10 (0.53; 2.2) 0.0–15 (0.75; 3.2)

0.0–130 (1.2; 38) 0.0–80 (1.4; 40) 0.0–2.6 (0.35; 1.9) 0.0–2.9 (0.35; 1.9) 0.15–8.7 (0.96; 4.9) 0.08–5.3 (1.0; 4.4) 0.0–0.46 (0.05; 0.40) 0.08–5.3 (1.0; 4.4) 0.14–14 (0.86; 8.5) 0.06–17 (0.91; 8.7)

–

TDI (EFSA)b Range (median; P95)

Senior citizens (n = 69/71)

Range (median; P95)

Adults (n = 267/269) Children II (n = 214/219) Children I (n = 30/31) Phthalate Model

Range (median; P95)

Range (median; P95)

Acceptable exposure levels (␮g/kg bw/d)

RfD AAd

C. Hartmann et al. / International Journal of Hygiene and Environmental Health 218 (2015) 489–499 Table 5 Daily phthalate intakes (ranges, medians, 95th percentiles) (␮g/kg bw/d) estimated with the creatinine-based calculation model among the study groups Children I (n = 30), Children II (n = 214), Adults (n = 267) and Senior citizens (n = 69), and with the volume-based calculation model among the groups Children I (n = 31), Children II (n = 219), Adults (n = 269) and Senior citizens (n = 71) and Tolerable Daily Intakes (TDI), Reference Doses (RfD) and Reference Doses for Anti-Androgenicity (RfD AA) (␮g/kg bw/d) of investigated phthalates.

494

90.0 80.0 70.0 Children I

60.0

Children II

50.0

Adults

40.0

Senior citizens

30.0 20.0 10.0 0.0 0.01

0.1

1

10

100

estimated creatinine-based daily DEHP intake (µg/kg bw/d) Fig. 1. Relative cumulative frequencies for the estimated creatinine-based daily DEHP intake in Children I (n = 30), Children II (n = 214), adults (n = 267) and Senior citizens (n = 69).

(median 0.9 ␮g/kg bw/d, P95 11 ␮g/kg bw/d) and senior citizens (median 1.2 ␮g/kg bw/d; P95 38 ␮g/kg bw/d). Results were similar for the volume-based calculation model. Fig. 1 represents the distribution plot of creatinine-based daily DEHP intakes (logarithmic scale for the x-axis) against the relative cumulative frequency distribution, illustrating the higher DEHP intakes in children compared to adults and senior citizens. For the study population groups Children II, Adults and Senior citizens, the curves have roughly the same slope. Across all study populations, median DnBP intakes were within the range of 0.24 to 0.84 ␮g/kg bw/d and DiBP intakes were within the range of 0.78 to 2.3 ␮g/kg bw/d, again with higher values for children compared to adults. Median BBzP daily intakes were lowest, ranging from 0.04 to 0.21 ␮g/kg bw/d. Calculated creatinine- and volume-based daily phthalate intakes were compared with results from several other European studies as summarized in Table 6. Thus far, only a few studies on daily phthalate intakes based on urinary metabolite levels are available. The majority of studies investigating European populations have been performed in Germany and Denmark. Compared to the estimated median daily phthalate intakes of German children aged 2–14 years from the German Environmental Survey IV (GerES IV) pilot study (Koch et al., 2007; Wittassek et al., 2007a), and of Danish children aged 6–11 years from urban areas (Frederiksen et al., 2013), median daily intakes of almost all investigated phthalates were lower in children and adolescents aged 6–15 years from the present study. Results for DEP were similar to those from Denmark. A study of German primary school starters (age 5–6 years) examined daily intakes of several phthalates (Koch et al., 2011) and demonstrated results comparable to those from this study conducted on children aged 6–8 years. Median daily intakes of DnBP, BBzP and DEHP were higher in German children, whereas the daily DiBP intake was found to be higher in this study population. Investigations of Danish women (age 31–52) from urban and rural areas, respectively (Frederiksen et al., 2013) showed slightly lower median daily intakes of DEP, DiBP and DEHP, and slightly higher daily intakes of DnBP and BBzP compared to those of the women from the present study aged 18–65 years. The results of a study conducted on German male and female adults aged 18–40 years (Koch et al., 2003a) showed extremely higher median daily phthalate intakes than those of the comparable population group as derived from our study. The largest difference was found for median daily DEHP intake in males, which was about 47 times higher in the German population. However, the different sampling years must be taken into account, since samples from Germany were taken in 2002, which may be one reason for these discrepancies. Estimated daily phthalate intakes calculated with both models (volume-based and creatinine-based) were compared with the

Table 6 Estimated daily phthalate intakes (ranges, medians, 95th percentiles) (␮g(kg bw/d) from selected studies in European populations. Study Germany Koch et al. (2007), and Wittassek et al. (2007a)

Koch et al. (2003a)

Koch et al. (2011) Fromme et al. (2013a) Kasper-Sonnenberg et al. (2014) Denmark Frederiksen et al. (2011) Bekö et al. (2013) Frederiksen et al. (2013)

France Zeman et al. (2013) Austria This study

a b c d e f g h i j k l

n

Sampling year

Calculation model

Male and female children (2–14 years) Male and female children (2–14 years) Male adults (18–40 years)

239

2001–2002

Creatinine-baseda

2001–2002

Volume-baseda

25

2002

Creatinine-basedb

Female adults (18–40 years)

34

2002

Creatinine-basedb

Male and female students

59

Volume-basedd

1.9; 5.3

108

2007

Creatinine-basede

2009–2010

Volume-basedf

465

2009–2010

Volume-basedg

Male and female children and adolescents (6–21 years) Male and female children (3–6 years) Male and female children from urban areas (6–11 years) Male and female children from rural areas (6–11 years)

129

2006–2008

Volume-basedh

1.1; 8.0

431

2008–2009

Volume-basedi

0.62; 3.9

3.3; 10.0

74

2011

Creatinine-basedj

0.53; 3.0

0.70; 2.2

67

2011

Creatinine-basedj

0.53; 2.7

0.86; 2.0

Pregnant women

139–279

2007

creatinine-basedk

1.0; 20.2

Male and female children (6–15 years) Male and female children (6–15 years) Male and female adults (18–81 years) Male and female adults (18–81 years)

250

2010–2011

Volume-basedl

0.69; 4.5

0.44; 1.7

2010–2011

Creatinine-basedl

0.60; 3.8

2010–2011

Volume-basedl

2010–2011

Creatinine-basedl

Male and female primary school starters (5–6 years) Male and female infants (1.25–1.75 years) Male and female children (8–10 years)

239

25

244 340 336

DEP Median; P95

DnBP Median; P95

DiBP Median; P95

BBzP Median; P95

DEHP Median; P95

4.1; 14.9

0.42; 2.6

4.3; 15.2

7.6; 30.5

0.77; 4.5

7.8; 25.2

2.4; 20.0

6.0; 19.9

1.1; 4.1

6.4; 24.4c

4.4; 33.6

8.1; 24.1

1.4; 5.0

4.7; 10.3c

1.4; 3.9

0.22; 0.91

2.4; 5.7

1.9; 6.4

2.1; 11.0

0.3; 2.6

4.5; 18.0

0.06; 0.14

0.43; 0.68

0.53; 1.1

0.95; 4.5

1.82; 5.86

2.18; 9.65

2.6; 4.0 0.23; 1.3

1.31; 4.31

0.62; 3.78

4.0; 10.7

0.49; 2.8

4.4; 16.9

2.4; 7.6

0.17; 1.1

2.7; 8.1

2.8; 7.4

0.23; 1.1

2.4; 12.5

0.4; 2.4

5.8; 65.1

1.4; 5.4

0.10; 0.84

1.6; 7.0

0.38; 2.2

1.1; 8.2

0.09; 0.92

1.5; 7.8

1.3; 15.3

0.29; 1.5

1.0; 4.4

0.05; 0.39

0.78; 3.4

0.95; 17.1

0.26; 1.3

0.8; 3.5

0.04; 0.24

0.59; 2.9

1.5; 6.6

2.2; 11.1

FUE adopted from Koch et al. (2005). FUE adopted from Anderson et al. (2001) for DnBP and BBzP. Corrected with metabolic conversion factors from Koch et al. (2005). FUE adopted from Anderson et al. (2001) for DnBP, DiBP, and BBzP, and from Koch et al. (2005) for DEHP. FUE adopted from Koch et al. (2005) for DEHP, and from Koch and Calafat (2009) and Wittassek et al. (2011) for DnBP, DiBP, and BBzP. FUE adopted from Anderson et al. (2001) for DEP and BBzP, from Seckin et al. (2009) for DnBP, and from Anderson et al. (2001), Kessler et al. (2012) and Koch et al. (2012) for DEHP related to the specific metabolite. FUE adopted from Anderson et al. (2001) and Koch et al. (2005, 2007). FUE adopted from Anderson et al. (2011) and Koch et al. (2005, 2007) for BBzP and DEHP, and from Koch and Calafat (2009) for DEP. FUE adopted from Koch et al. (2012) for DiBP. FUE adopted from Anderson et al. (2001, 2011) for DEHP and BBzP, and from Koch and Calafat (2009) for DEP. FUE adopted from Koch et al. (2005) for DEHP, from Anderson et al. (2001) for DnBP, from Fromme et al. (2007) for DEP and DiBP, and from Koch et al. (2003b) for BBzP. FUE adopted from Anderson et al. (2011) for DEHP, from Koch and Angerer (2012) for DEP, from Koch et al. (2012) for DnBP and DiBP, and from Anderson et al. (2011) for BBzP.

C. Hartmann et al. / International Journal of Hygiene and Environmental Health 218 (2015) 489–499

Wittassek et al. (2007b)

Population

495

496

C. Hartmann et al. / International Journal of Hygiene and Environmental Health 218 (2015) 489–499

Table 7 Hazard Indices (HI) for Austrian children, adults and senior citizens based on tolerable daily intake (TDI) and reference dose for anti-androgenicity (RfD AA), respectively.

n HI (TDI); n > 1 (%) HI (RfD AA); n > 1 (%)

Children I range (median; P95)

Children II range (median; P95)

Adults range (median; P95)

Senior citizens range (median; P95)

30 0.08–3.4 (0.37; 2.7); 13.3 0.02–0.74 (0.13; 0.69); 0

214 0.02–5.4 (0.18; 0.94); 4.2 0.01–0.72 (0.05; 0.28); 0

266 0.0–1.7 (0.12; 0.42); 0.4 0.0–0.35 (0.27; 0.09); 0

69 0.02–1.1 (0.15; 0.77); 2.9 0.01–0.48 (0.04; 0.31); 0

HI, Hazard Index; n, sample size; RfD AA, Reference Dose for Anti-Androgenicity; P95, 95th percentile. Notes: Hazard Indices calculations based on DnBP, DiBP, BBzP and DEHP related to the RfD AA, and on DnBP, DiBP and DEHP related to the TDI because of the same endpoints (anti-androgenicity).

existing acceptable exposure levels (AL) listed in Table 5 and comprising the Tolerable Daily Intake (TDI), the Reference Dose (RfD) and the Reference Dose for Anti-Androgenicity (RfD AA) established by EFSA, U.S. EPA and Kortenkamp and Faust (2010), respectively. As claimed before, misclassifications of exposure cannot be completely excluded and interpretation of individual exposure levels have to be handled with care due to the use of spontaneous urine samples and the rapid elimination half-times of phthalates. Based on the creatinine calculation model, one child from the Children I group (out of 30) and one child from the Children II group (out of 214) exceeded the EFSA TDI for DnBP of 10 ␮g/kg bw/d (daily DnBP intakes of 14.7 and 18.6 ␮g/kg bw/d, respectively). Calculated creatinine-based daily DiBP intakes of four children from Children I (ranging between 11.3 and 16.7 ␮g/kg bw/d), four children from Children II (16.6 to 34.3 ␮g/kg bw/d) and one adult (out of 267; 16.0 ␮g/kg bw/d) exceeded the EFSA TDI of 10 ␮g/kg bw/d. Additionally, the P95 value exceeds the TDI among the Children I group. Based on the volume calculation model, one adult (out of 269) also exceeded the TDI. For creatinine-based daily DEHP intake, one exceedance of the U.S. EPA RfD of 20 ␮g/kg bw/d was identified among the Children II group (21.1 ␮g/kg bw/d). No exceedances existed for the volume- and creatinine-based daily intakes of DEP and BBzP.

Cumulative risk assessment Usually, risk assessment of chemicals does not account for effects of combined exposures, which may lead to underestimations of risks. Thus, cumulative risk assessment considering effects of exposure to several chemicals through various routes and pathways is needed. One approach that allows the assumption of adverse effects to human health resulting from simultaneous exposures is the dose-addition concept by using the Hazard Index (HI) for the estimation of the overall potential for non-carcinogenic effects of several chemicals (Christensen et al., 2014; Kortenkamp and Faust, 2010; Søeborg et al., 2012; U.S. EPA (United States Environmental Protection Agency), 1989). Based on the calculated daily intakes derived from urinary metabolite levels and acceptable levels of exposure (AL) for each individual phthalate, a cumulative risk assessment was performed using equation (3) for the antiandrogenic phthalates DEHP, DnBP, DiBP and BBzP related to the Reference Dose for Anti-Androgenicity (RfD AA) as well as for DEHP, DnBP and DiBP related to the Tolerable Daily Intake (TDI). For DEHP and DnBP, the TDIs are based on peroxisome proliferation in rodent liver (EFSA (European Food Safety Authority), 2005a, 2005b, 2005c). The TDI of DnBP was used in analogy for DiBP, because of the similarity of DiBP to DnBP in relation to effects on development and reproduction (EC (European Commission), 2013). For cumulative risk assessment, the RfDs established by the U.S. EPA are not suitable, because they are based on other endpoints than antiandrogenicity (U.S. EPA (United States Environmental Protection Agency), 2007a,b). As shown in Table 7, median HIs based on all ALs used are far below the value of 1. The highest values were identified among children, where samples from the Children I group showed higher

values than those of the Children II group. Exceedances of the HI of 1 existed among all age groups for TDI based values. Four out of 30 children from Children I (13%), 9 out of 214 children from Children II (4%), 1 out of 266 adults (0.4%) and 2 out of 69 senior citizens (3%) exhibited an exceedance of a HI of 1, with the maximum HI being identified in the Children I group (2.7). Based on calculations related to RfD AA, no exceedances were identified. This is mainly due to the underlying RfD AA values being up to 20 times higher (except for DEHP) than the underlying TDI values. Currently, only a few previous studies on hazard indices exist. In 129 Danish children and adolescents, 19 children exceeded the HI based on EFSA TDI and one child exceeded the HI based on RfD AA (Søeborg et al., 2012). In young children from Germany (5–6 years), Koch et al. (2011) evaluated the relative tolerable daily intake (TDIcum ) based on DnBP, DiBP and DEHP, which is comparable with the hazard index approach. 26 out of 108 children exceed the TDIcum . Dewalque et al. (2014) reported in a study of 52 male and female children (1–12 years) and 209 adults (13–85 years) from Belgium exceedances of the HI based on TDIs of 25% in children and 6.2% in adults. In 33 Danish men aged 18–22 years, three 24-h urine samples were collected from each participant. Based on the EFSA TDI, two men exceeded the related TDI, and based on RfD AA, one man exceeded the related HI (Kranich et al., 2014). When compared to results derived from German children (Koch et al., 2011), slightly fewer exceedances of TDI-based HIs existed in our study. However, it should be taken into account that the sample size was smaller in the present study. Compared to Danish children (Søeborg et al., 2012), the number of HI exceedances is similar. In adult men, exceedances of HI based on TDI and RfD AA, respectively, were more frequent in Danish participants. In comparison with Belgian children and adults (Dewalque et al., 2014), exceedances of HI identified in our study were lower, especially among adults.

Conclusion With the present study we provide first human biomonitoring derived phthalate exposure data for a subset of the Austrian population including children from the age of 6 to senior citizens. In many ways, phthalate exposure in Austria is similar to that of other European countries or the situation worldwide with (younger) children exhibiting the highest levels of exposure to most of the anti-androgenic phthalates. We also found a tendency of higher exposures to several phthalates among the female population. Regarding geographical differences, statistically significant higher levels of exposure to several phthalates were demonstrated in participants from (sub)urban areas compared to those from rural areas. Most of the above findings might be explained by age- and sex-specific behaviours, lifestyles and differences in the use of consumer products, cosmetics and personal care products. For the individual phthalates, and after the calculation of the daily intakes, we observed only a few exceedances of TDI values for DiBP (n = 4), DnBP (n = 2) and DEHP (n = 1), mainly among the youngest children. The daily intakes we calculated for our subset of the Austrian population were generally lower than those of other European countries or of the rest of the world. Possible explanations

C. Hartmann et al. / International Journal of Hygiene and Environmental Health 218 (2015) 489–499

for these differences include differences in nutrition and lifestyle, but also differences in the years of sampling. Our samples were collected in 2011 and might already reflect the decreasing use of DnBP and DEHP in Europe due to regulatory restrictions. The execution of a cumulative risk assessment for combined phthalate exposure demonstrated exceedances of the HI based on TDIs of 13.3% of the children from the Children I group (6–8 years) and of 4.2% of the children from the Children II group (7–15 years), which might constitute a risk of anti-androgenic effects during puberty, which is a sensitive stage regarding hormonal changes and the development of reproductive organs. Although exposure seems to be lower in the Austrian population, phthalates are widely distributed, especially among children, who represent a vulnerable group, and showed few but existing exceedances of acceptable exposure levels such as the EFSA TDI. Taking into consideration that children are exposed to various other chemicals such as pesticide residues and certain ingredients of cosmetics (e.g. parabens and UV filter substances) which also act anti-androgenically, there is potential indication of cause for concern. In the present study, the investigation of the exposure to secondary metabolites of the phthalates DiNP and DiBP have not yet been included. Because of their importance, especially regarding DiNP exposure, measurements of these metabolites will be included in future. Acknowledgement Parts of the study were financed by the Austrian Federal Ministry of Agriculture, Forestry, Environment and Water Management and by the Austrian Environment Agency. Samples were provided from the Austrian Study of Nutritional Status 2012 (ASNS) which was funded by the Austrian Federal Ministry of Health. We want to thank Prof. Dr. Ibrahim Elmadfa for the possibility for ASNS cooperation. C. Hartmann was recipient of a DOC-fFORTE Fellowship of the Austrian Academy of Sciences at the Institute of Nutritional Sciences of the University of Vienna. References Anderson, W.A.C., Castle, L., Scotter, M.J., Massey, R.C., Springall, C., 2001. A biomarker approach to measuring human dietary exposure to certain phthalate diesters. Food Addit. Contam. 18, 1068–1074. Anderson, W.A.C., Castle, L., Hird, S., Jeffery, J., Scotter, M.J., 2011. A twenty-volunteer study using deuterium labelling to determine the kinetics and fractional excretion of primary and secondary urinary metabolites of di-2-ethylhexylphthalate and di-iso-nonylphthalate. Food Chem. Toxicol. 49, 2022–2029. Bekö, G., Weschler, C.J., Langer, S., Callesen, M., Toftum, J., Clausen, G., 2013. Children’s phthalate intakes and resultant cumulative exposures estimated from urine compared with estimates from dust ingestion, inhalation and dermal absorption in their homes and daycare centers. PLoS ONE 8 (4), 1–18 (e62442). Bornehag, C.G., Sundell, J., Weschler, C.J., Sigsgaard, T., Lundgren, B., Hasselgren, M., Hägerhed-Engman, L., 2004. The association between asthma and allergic symptoms in children and phthalates in house dust: a nested case-control study. Environ. Health Perspect. 112, 1393–1397. Bornehag, C.G., Nanberg, E., 2010. Phthalate exposure and asthma in children. Int. J. Androl. 33, 333–345. Braun, J.M., Sathyanarayana, S., Hauser, R., 2013. Phthalate exposure and children’s health. Curr. Opin. Pediatr. 25 (2), 247–254. CDC (Center of Disease Control Prevention), 2014. Fourth National Report on Human Exposure to Environmental Chemicals, Updated Tables, August 2014. Centers for Disease Control and Prevention, Atlanta, GA, USA, http://www. cdc.gov/exposurereport/pdf/FourthReport UpdatedTables Aug2014.pdf. Christensen, K.L.Y., Makris, S.L., Lorber, M., 2014. Generation of hazard indices for cumulative exposure to phthalates for use in cumulative risk assessment. Regul. Toxicol. Pharm. 69, 380–389. Danish EPA (Danish Environmental Protection Agency), 2011. Annex XV Restriction Report, Proposal for a Restriction, Substance Name: bis(2-Ethylhexyl)phthalate (DEHP), Benzyl Butyl Phthalate (BBP), Dibutyl phthalate (DBP), Diisobutyl phthalate (DIBP), Version Number 2. Danish Competent Authority for REACH, Copenhagen, Denmark. Danish EPA (Danish Environmental Protection Agency), 2013. Phthalate Strategy. Danish Environmental Protection Agency, Copenhagen, Denmark.

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