Bioavailability of phthalate and DINCH® plasticizers, after oral administration of dust to piglets

Toxicology Letters 314 (2019) 82–88

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Bioavailability of phthalate and DINCH® plasticizers, after oral administration of dust to piglets

T

V. Plichtaa, W. Völkela, , L. Fembachera, M. Spoldersb, M. Wöcknera, B. Aschenbrennera, H. Schafftb, H. Frommea,c ⁎

a

Bavarian Health and Food Safety Authority, Department of Chemical Safety and Toxicology, D-80538, Munich, Germany German Federal Institute for Risk Assessment, D-10589, Berlin, Germany c Institute and Outpatient Clinic for Occupational, Social and Environmental Medicine, Ludwig-Maximilians-University, D-80336, Munich, Germany b

ARTICLE INFO

ABSTRACT

Keywords: Dust Phthalates DINCH® Oral bioavailability Exposure

For decades, phthalates have been widely used as plasticizers in a large number of consumer products, leading to a complex exposure to humans via ingestion, inhalation or dermal uptake. Children may have a higher unintended dust intake per day compared to adults. Therefore, dust intake of children could pose a relevant exposure and subsequently a potential health risk. The aim of this study was to determine the relative bioavailability of certain phthalates, such as di(2ethylhexyl) phthalate (DEHP), di-isononyl phthalate (DINP) and the non-phthalate plasticizer diisononyl 1,2cyclohexanedicarboxylic acid (DINCH®, Hexamoll®), after ingestion of dust. Seven 5-week-old male piglets were fed five different dust samples collected from daycare centers. Overall, 0.43 g to 0.83 g of dust sieved to 63 μm were administered orally. The piglets’ urine was collected over a period of 38 h. The excreted metabolites were quantified using an LC–MS/MS method. The mean uptake rates of the applied doses for DEHP, DINP, and DINCH® were 43% ± 11%, 47% ± 26%, and 9% ± 3.5%, respectively. The metabolites of DEHP and DINP showed maximum concentrations in urine after three to five hours, whereas the metabolites of DINCH®, reached maximum concentrations 24 h post-dose. The oral bioavailability of the investigated plasticizers was higher compared to the bioaccessibility reported from in vitro digestion tests. Furthermore, the bioavailability of DEHP did not vary substantially between the dust samples, whereas a dose-dependent saturation process for DINP was observed. In addition to other intake pathways, dust could be a source of plasticizers in children using the recent intake rates for dust ingestion.

1. Introduction In industrial countries, humans spend approximately 90% of their time indoors, either at home, in offices or in transport facilities. Analyzing indoor dust can provide information regarding the presence of and exposure to pollutants in indoor environments. House dust is a mix of different contaminants and acts as a transport medium for allergens, heavy metals, and semi and non-volatile chemicals, e.g., phthalates and other plasticizers (Molhave et al., 2000; Huwe et al., 2008; Cizdziel and Hodge, 2000; US General Accounting Office, 1999; World Health Organisation, 1999; US-Environmental Protection Agency, 2011). The US-EPA has estimated the average daily intake between 20–50 mg dust. Babies (< 6 months) and adults (> 12 years) have an unintended daily intake of 20 mg of dust, while toddlers (1–2 years) and children (2–12 years) ingest approximately 50 mg and 30 mg

⁎

daily, respectively (US-Environmental Protection Agency, 2017). Because of their hand to mouth contact and frequent object to mouth behavior, children ingest more dust than adults, which might be related to potential health risks (Moya and Phillips, 2014). As a consequence of their ubiquitous usage, phthalates are one of the most frequent occurring compounds in house dust (Blanchard et al., 2014). Phthalates are widely used as plasticizers to increase the flexibility and softness of a wide variety of plastic products and are added to other materials, such as personal care products, children’s toys, building materials and food packaging (Gimeno et al., 2014; Rudel et al., 2003; Australian Government Department of Health and Ageing, N., 2008). Phthalates are endocrine disrupters and primarily target the male reproduction system. Humans are exposed to phthalates by ingestion, inhalation or dermal uptake. In the body, these substances undergo a rapid metabolism, and the formed metabolites are excreted

Corresponding author at: Bavarian Health and Food Safety Authority, Department of Chemical Safety and Toxicology, D-80538, Munich, Germany. E-mail address: [email protected] (W. Völkel).

https://doi.org/10.1016/j.toxlet.2019.07.018 Received 22 February 2019; Received in revised form 23 June 2019; Accepted 1 July 2019 Available online 12 July 2019 0378-4274/ © 2019 Elsevier B.V. All rights reserved.

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with urine within two days. The phthalates used in high quantities such as di-2-ethylhexyl phthalate (DEHP) have been classified as reprotoxic and often been replaced by diisonyl phthalate (DINP) (European Commission, 2003) and the non-phthalate plasticizer diisononyl 1,2cyclohexanedicarboxylic acid (DINCH®, Hexamoll®). In 2002, DINCH® has been introduced to the market, because of its lower migration rate and lower toxicity (European Food Safety Authority, 2006). There are several studies that have investigated phthalates in indoor dust, with concentrations of DEHP, DINP, and DINCH®, varying from 9.9 to 10.1 mg/kg, 12 to 7091 mg/kg, and 32 to 2732 mg/kg, respectively (Molhave et al., 2000; Blanchard et al., 2014; Abb et al., 2009; Ait Bamai et al., 2014; Bornehag et al., 2005; Fromme et al., 2013a; Lioy et al., 2002; Orecchio et al., 2013; Wei et al., 2009; Fromme et al., 2016). Depending on its concentration in dust, the mean oral DINP and DEHP exposure through dust ingestion in children ranges between 1.2 and 21 μg/kg per day (Oomen et al., 2008). Several biomonitoring studies have investigated the correlation between children’s phthalate exposure and the concentration found in dust samples, whereas for DINCH® only a limited number of studies can be found (Fromme et al., 2013a; Fromme et al., 2016; Becker et al., 2004; Beko et al., 2015). Several studies have indicated that phthalate exposure through dust contributes significantly to the total DEHP body burden (Fromme et al., 2013a, 2004; Guo and Kannan, 2011), whereas other studies could not observe a significant proportion (Becker et al., 2004; Langer et al., 2014). Currently, there is no information available about the oral bioavailability of phthalates in dust to humans or experimental animals. The oral bioavailability is the fraction of an ingested contaminant in a certain carrier matrix that reaches systemic circulation. It is influenced by at least three factors, first the release of the contaminant from the carrier matrix in the gastro-intestinal tract, secondly the absorption rate and thirdly the metabolism of the contaminant in the intestine and liver (Brandon et al., 2006). Up to now only two studies investigated the oral bioaccessibility of phthalates in house dust by an in vitro digestion test. Both studies showed that the bioaccessibility ranged between 2% and 15%, depending on the type of phthalate (Kang et al., 2012; Wang et al., 2013). Therefore, it is still a matter of scientific debate regarding how much dust intake contributes to overall plasticizers exposure. The goal of this study was to examine the oral bioavailability of plasticizers in house dust after ingestion using piglets as a model. We focused on the plasticizers DEHP, DINP, and DINCH®, which are present in dust to a large extent and for which biomonitoring studies have shown that humans are highly exposed. Another aim of the study was to determine if dust is a relevant source of phthalates and DINCH® exposure, especially for children.

Table 1 Overview of phthalate concentration in the administered dust sample in mg/kg.

DEHP DINP DINCH®

Dust 1

Dust 2

Dust 3

Dust 4

Dust 5

6900 1100 1100

8700 470 2000

7000 1700 610

5800 2100 690

4800 4300 1300

concentrations of the plasticizers in dust are summarized in Table 1. The applied dose oriented to the tolerable daily intake (TDI) of DEHP (50 μg/kg b.w.) estimated by EFSA (European Food Safety Authority, 2005). The order of dust samples varied and before, during and after an experiment any contact with any plasticizers was avoided. After each experiment, a “washout “period of three days was applied without any known phthalate exposure to avoid any influence from the previous experiment. To evaluate the background exposure, a urine sample (control urine) was collected before the dust administration. The urine was collected into polyethylene (PE) specimen containers (Sarsted, Nümbrecht, Germany) at the following time points: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 24, 28, 32, 36 and 38 h after dust administration. The volume of the collected urine was noted and an aliquot of approximately 100 ml of the excreted sample was placed in another PE cup and stored at −20 °C until further analysis. During the experiment, the piglets stayed in a metabolic cage, and during their three-day wash out period, two piglets each shared a daylight pigpen, and the floor was covered with straw. In general, the piglets had access to water ad libitum and received a diet twice a day (900 g cooked peeled potatoes). The phthalate concentrations in the feed were measured, as well as in the indoor air of the pigpen. In the feed samples, the analysed concentrations were below the limit of detection (LOD) of 5 ng/g. In the air of only one pigpen DEHP was detected at a concentration of 55 ng/m³, while the other phthalates were below the LOD of 50 ng/m³. 2.2. Analytical methods 2.2.1. Analysis of phthalates and DINCH® in dust samples The dust samples originated from a previously performed study in different daycare centers in Germany (Fromme et al., 2013a). In that study, the dust samples were obtained from the vacuum bags of vacuum cleaners in regular use and were sieved to 63 μm. Overall, 100–150 mg of the sieved dust was spiked with internal standard DEHP-d4. After adding 10 ml of methyl-tert-butyl-ether (MTBE), the samples were sonicated (15 min) and subsequently transferred to centrifuge tubes and centrifuged (3076 g, 5 min, 5 °C). The supernatant was placed in a brown glass screw cap vial. The residue was dissolved again with 5 ml of MTBE for a second extraction and centrifugation was performed as described above. The samples, including a control blank, were analyzed by a gas chromatography system with a mass selective detector in electron impact (EI) mode (Shimadzu GC–MS QP2010, Duisburg, Germany) with a 30 m/0.25 mm, ID/0.25 μm Phenomenex Zebron ZB-5 ms column (Phenomenex, Aschaffenburg, Germany). A detailed description of the method has already been given in (Fromme et al., 2013a). For quality control, a reference dust (NIST standard-reference material SRM 2585: Organic Contaminants in house dust) was measured. The recovery was between 80 and 100% for all analytes.

2. Materials and methods 2.1. Study design Seven 5- to 6-week-old male piglets with a body weight of 11 kg–14 kg received five different dust samples. Piglets were chosen as a model organism because of the similarity in the metabolism rate and the physiological properties in the gastrointestinal tract to humans. For practical reasons as e.g. collecting the urine, we decided to take only male piglets. As described in previous studies (Koch et al., 2013a; Koch and Angerer, 2007; Koch et al., 2005) urine is the main route of phthalate and DINCH® excretion, therefore we collect urine. The experiment took place at the German Federal Institute for Risk Assessment (BfR) in Berlin, Germany and was approved by the Regional Office of Health and Social Affairs, Berlin, Germany (LAGeSo, Reg 0272/13). The experiment plan appears in the supplementary materials in Table S1. During an experiment one dust samples was mixed with the morning feedings and orally administered to the piglets. The amount of given dust ranged from 432 mg to 832 mg (for details see Table S2). The

2.2.2. Analysis of plasticizer metabolites in urine The quantitation of plasticizer in urine samples were performed with MultiQuant 2.1.1. (AB SCIEX). In general, the quality control samples were in good agreement with the expected concentration of 20 pg/μl (recovery was 80–100%). The variation of the duplicate urine samples was below 15%. 2.2.2.1. Chemicals. The native standards mono(2-ethylhexyl) phthalate (MEHP), mono(2-ethyl-5-hydroxyhexyl) phthalate (5-OH-MEHP), 83

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mono(2-ethyl-5-oxohexyl) phthalate (5-oxo-MEHP), mono-isononyl phthalate (MINP) and the labeled internal standards of 13C4-MEHP, 13C4-5-oxo-MEHP, and 13C4-MINP were purchased from Cambridge Isotope Laboratories (Andover, MA). The native standards mono(2-ethyl-5-carboxypentyl) phthalate (5cx-MEPP), mono(4-methyl-7-hydroxy-octyl) phthalate (7-OH-MINP), mono(4-methyl-7-carboxy-heptyl) phthalate (7-cx-MINP) and the labeled internal standards: D4-5-OH-MEHP, D4-5-cx-MEPP, D4-7-OHMiNP, and D4-7-cx-MINP were purchased from the Department of Biochemistry, Institute of Environmental Carcinogens (Germany). Cyclohexane-1,2-dicarboxylate-mono-4-methyloctyl ester (MINCH), cyclohexane-1,2-dicarboxylate- mono-(7-carboxylate-4-methyl) heptyl ester (cx-MINCH), cyclohexane-1,2-dicarboxylate-mono-(7-hydroxy-4methyl) octyl ester (OH-MINCH) and their side-chain labeled analogous structures cyclohexane-1,2-dicarboxylate-mono- D2-4-methyloctyl ester (D2-MINCH), cyclohexane-1,2-dicarboxylate-mono-D2-(7-carboxylate-4-methyl) heptyl ester (D2-cx-MINCH), cyclohexane-1,2-dicarboxylate-mono- D4-(7-hydroxy-4-methyl) octyl ester (D4-OHMINCH) were a kind gift from Koch from Institute for Prevention and Occupational Medicine of the German Social Accident Insurance, Institute of the Ruhr-University Bochum (IPA), Germany. Acetonitrile (HPLC grade) was purchased from Fischer Chemicals, Germany). Acetic acid and formic acid were obtain from Carl Roth (Germany). Ammonium acetate was purchased from Riedl-de Haën and β-glucuronidase (Type 2 H-2 Helix pomatia) were purchased from Sigma–Aldrich (Germany).

chromatographic separation, an Atlantis dC18 (2.1 x 150 mm; 3 μm) (Waters, Eschborn, Germany). The LOQ were 0.1 μg/l for monoisononyl ester (MINCH) and 0.05 μg/l for the secondary oxidized metabolites OH-MINCH, oxo-MINCH, and cx-MINCH.

2.2.2.2. Analysis of phthalate metabolites in urine. The metabolites were analyzed in urine after enzymatic hydrolyses via HPLC-MS/MS. The method has already been described in detail by Fromme et al. (2013b). In short, 200 μl of the thawed, room temperature urine samples were mixed with 55 μl of 1 M ammonium acetate (pH 6.5,), 10 ng of an internal standard mix (1 ng/μl) and 5 μl of β-glucuronidase (Enzymatic deconjugation was performed in a water bath at 37 °C for 1.5 h). After that, 250 μl of acetonitrile were added and then the samples were centrifuged for 15 min at 20,800 g. The sample solutions were then transferred into vials that were filled up with 480 μl of 0.5% formic acid. All urine samples were prepared as duplicates and for quality control spiked samples (980 μl of urine and 20 μl of native standard mix [1 ng/μl]) were used. The urine samples taken in the third and fourth hour after administration were measured undiluted and diluted using purified water at a ratio of 1:10. Next, 50 μl of the supernatant was injected into an UltiMate™ 3000 (Thermo Scientific, Dreieich, Germany) coupled with an AB Sciex QTrap 5500 tandem mass spectrometer from SCIEX (Darmstadt, Germany). We used a Waters Oasis HLB (25 μm, 20 x 2.1 mm ID) as a trap column (Waters, Eschborn, Germany) and a Phenomenex Luna Phenyl–Hexyl (3 μm, 150 x 3 mm) as an analytical column (Phenomenex, Aschaffenburg, Germany). For quantification of the phthalate metabolites, a calibration curve with a known amount of metabolite concentration (0.5, 1, 2, 5, 10, 20, 50, 100, 200 and 400 pg/μl) was used. The limits of detection (LOD) are given at the S/N ratio = 3. LODs were 0.5 μg/l for oxo-MINP, oxoMEHP, 5 cx-MEPP; 1 μg/l for 7 OH-MINP, 5 OH-MEHP, MEHP; 1.3 μg/l for 7 cx-MINP; 1.5 μg/l for MINP; and 5 μg/l for 2cx-MMHPP.

t ½ = Ln (2)*ke

2.3. Statistical analysis For calculations and statistics, Microsoft Excel 2010 and SPSS 19 (IBM) were used. 2.3.1. Calculation of the plasticizers uptake using urinary metabolite concentration To estimate the oral bioavailability we calculated the intake [%] using the following equation:

recovery as urine metabolites [%] excreted metabolite / MW metabolite = x100 Amount of parentcompound / MW parentcompound

(1)

The following molecular weights (MW) [g/mol] were used: DEHP (390.56), MEHP (278.34), 5OH-MEHP (294.34), oxo-MEHP (292.32), 5cx-MEPP (308.30), 2cx-MMHP (308.32), DINP (418.62), MINP (292.38), oxo-MINP (306.35), 7OH-MINP (308.37), 7cx-MINP (322.35), DINCH® (424.7), MINCH (298.42), oxo-MINCH (312.4), OHMINCH (314.42), cx-MINCH (328.4). The elimination half-life time (t½) of the metabolites was calculated using the following equation: where ke is the elimination rate constant, which was calculated by the excreted urinary concentration per unit time and the excreted urinary volume. The phthalate exposure through dust was calculated as followed:

Exposure Plasticiziser (concentration of plasticizers in dustxaverage daily dust uptake ) x1000 = bodyweight of the child (2) 3. Results and discussion In most instances, no metabolites were detected in the control urine samples, or only small amounts (< 1.5 μg/l). The mean administered dose of plasticizers in the dust ranged from 219 μg (DINP) to 4057 μg (DEHP), respectively (see Table 2). Using Eq. (1), the sum of the mean recovery as urine metabolites was 43% ± 11% for DEHP, 47% ± 26% for DINP, and 9% ± 3.5% for DINCH® for the applied dust samples. In more detail, the mean excretion of DEHP, DINP and DINCH® for dust sample 1 was 44% ± 8.5%,75% ± 21%, and 8% ± 2%; for dust sample 2: 42% ± 14% (DEHP), 8% ± 3% (DINCH®); for dust sample 3: 46% ± 16% (DEHP), 52% ± 28% (DINP), 10% ± 4% (DINCH®); for dust sample 4: 39% ± 6% (DEHP), 37% ± 9% (DINP), 13 ± 3% (DINCH®); and for dust sample 5: 44% ± 6% (DEHP), 23% ± 6% (DINP), 8% ± 2% (DINCH®) (see Fig. 1). Table S3 in the supplementary materials listed the percentage dose uptake for each dust sample and

2.2.2.3. DINCH® metabolite analysis in urine. Analyzing DINCH® metabolites in urine is similar to the phthalate method and has been described by (Schütze et al. (2012)). In brief, 300 μl of the urine samples were spiked with 100 μl 1 M ammonium acetate, 10 μl internal standard and 6 μl β-glucoronidase (diluted 1:1 with ammonium acetate), then deconjugated for 2 h at 37 °C. Next, 10 μl acetic acid was added to adjust the pH value and then the samples were centrifuged at 1900 g for 10 min, and 25 μl of the supernatant was injected into the same HPLC-MS/MS system as used for the phthalates metabolites. We used a Capell PAK 5 μm C18 MG-II (Phenomenex, Aschaffenburg, Germany) column for cleanup and enrichment and for

Table 2 The mean and standard deviation (SD) of the administered dose in μg.

Dust Dust Dust Dust Dust

84

1 2 3 4 5

DEHP

DINP

DINCH®

3989 ± 113 4057 ± 346 3874 ± 215 3878.5 ± 86 3728.5 ± 188

636 ± 18 219 ± 19 941 ± 52 1404 ± 31 3340 ± 168

636 ± 18 932 ± 80 337 ± 18.7 461 ± 10.3 1010 ± 51

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piglet. In the case of DINP in dust sample 2, all piglets excreted 133% to 315% of the administered dose. The piglets had no or minor background exposure of DINP and the dust samples were measured several times with similar results. Because of this, we rejected all DINP results from dust sample 2. Regional Office of Health and Social Affairs limits the collecting time to 38 h. We were still able to detect metabolites in the 38 h post dose urine samples and have to assume that the excretion of the applied plasticizers was not fully completed. However, as our data show, on the second day less than five percent of the dose were excreted and therefore only a minor amount of the complete doses was not accounted due to limited sampling time. The bioavailability of DEHP in the dust samples showed low variation and seems to have a good reproducibility (Fig. 1). Additionally, we found a saturation process: the higher the applied DINP dose, the lower the bioavailability. Fig. 1C shows that dust sample 1 with a mean concentration of 636 μg DINP has a remarkably high bioavailability of 75% ± 21% compared to the dust sample 5 with an applied dose of 3340 μg DINP and a bioavailability of 23% ± 5%. The applied amount of dust varies between 0.5 g (dust sample 1) and 0.78 g (dust sample 5). To clarify if the concentration or the amount of dust underlies a saturation process dust sample 1 and dust sample 3 were administered in a similar amount (0.54 g) and the concentration varies from 636 μg (dust sample 1) to 941 μg (dust sample 3). The bioavailability of DINP was determined for dust sample 1 75% ± 21% and for dust sample 3 52% ± 28%. Those results indicate that the saturation process depends on the applied concentration and not on the given amount of matrix. These findings confirm the results published by McKee et al. (2002),

who also observed a decrease of adsorption rate by an increasing orally administrated dose in rats. 49% of the given DINP dose (50 mg/kg) was determined, whereas there was only an uptake of 39% of the 500 mg/kg DINP dose. For DINCH® a dose-dependent systematic bioavailability was observed. Up to 50% of the lower dose (20 mg/kg bw/d) was excreted, while by a higher dose of 1000 mg/kg bw/d the bioavailability was by only 6% (Bhat et al., 2014). We did not observed this effect in DINCH® or other phthalates and further research for understanding this saturation process is needed. Our results suggest a higher bioavailability compared to the results of former in vitro studies. For example, in an in vitro digestion test, a bioaccessibility of 2.2%–12.6% for both DEHP and DINP in dust was observed (Wang et al., 2013). In another in vitro digestion test, slightly higher values were reported (10%–15% for both DEHP and DINP) (Kang et al., 2012). The different results between this study and the in vitro digestions tests indicate that the chosen parameters for the in vitro tests (pH, acid mixture, residual time, ratio between matrix and liquid) might not match the simulation of digestions, which could lead to an underestimation of the bioavailability. At the current state of knowledge in vitro digestions tests cannot accurately simulate the bioaccessibility of human digestions (in vivo) (Oomen et al., 2002). Thus, the results of an in vitro digestions test should considered as only indicative values. This study and both in vitro digestion test studies used dust particles sieved to 63 μm, where the highest bioavailability was expected. It has been shown under experimental conditions that phthalate accumulation strongly depend on particle size distribution in house dust. Wang et al. (2013) investigated the accumulation rate of phthalates in

Fig. 1. Percentage excretion of (A) DEHP (43%), DINP (47%) and DINCH (9%) after dust administration to all piglets, (B–D) the sum of (B) DEHP-(Dust 1: 44% ± 8.5%; Dust 2: 42% ± 14%; Dust 3: 45% ± 16%; Dust 4: 39% ± 6%; Dust 5: 44% ± 6%), (C) DINP- (Dust 1: 75% ± 21%; Dust 3: 52% ± 28%; Dust 4: 37% ± 9%; Dust 5: 23% ± 5%) and (D) DINCH®- metabolites (Dust 1: 8% ± 2.5%; Dust 2: 7.8% ± 3%; Dust 3: 9.7% ± 4%; Dust 4: 12.6% ± 3%; Dust 5: 8% ± 2%) in relation to the applied dose of dust. 85

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different particle size < 63 μm, 63–100 μm, 100–280 μm and 280 2000 μm; the highest rate were found at < 63 μm and 63–100 μm. The bioaccessibility decreased by increasing particle size. In a bioavailability study by Freeman et al. (1995) it was indicated that there is an association with the n-octanol/water coefficient (Kow) and the bioavailability. The higher the Kow value the lower the oral bioaccessibility. This could explain why the oral bioavailability of DINCH® (Kow 10) is lower compare to the phthalates (DEHP Kow: 7.5; DINP Kow 8,8) (Australian Government Department of Health and Ageing, N., 2008; BASF, 1999).

5.1 ± 2 h for 5cx-MEPP and 5.0 ± 1.8 h for 5OH-MEHP. Within the first 24 h, 40.2% ± 10.2% of the DEHP dose was excreted as 16.3% ± 4.6% 5cx-MEPP, 8.7% ± 3.8% 5OH-MEHP, 8.4% ± 2.6% oxo-MEHP, and 6.7% ± 3% MEHP. Between 24–38 hours, in total 2.7% ± 1% of DEHP dose was found in urine. It was mainly excreted as 1% ± 0.4% 5cx-MEPP followed by 0.7% ± 0.3% oxo-MEHP, 0.6% ± 0.4% 5OH-MEHP, and 0.4% ± 0.2% MEHP. To sum up, for all piglets administered the dust samples, the metabolites were excreted in the following order: MEHP (7% ± 3%) < oxo-MEHP (9.2% ± 3%) < 5OH-MEHP (9.4% ± 4%) < 5cx-MEPP (17.4% ± 5%).

3.1. Toxicokinetics of the plasticizers

3.1.2. Di-isononyl phthalate (DINP) The quantified DINP metabolites reached their tmax concentration 4.2 ± 4 h (MINP), 5.5 ± 6.6 h (oxo-MINP), 4.0 ± 4.2 h (7OH-MINP), and 4.8 ± 5.6 h (cx-MINP) after dose application (Fig. 2B). The estimated half-life time for the first elimination phase was quite similar for all metabolites with 5.3 ± 2 h for MINP, 6.6 ± 4 h for oxo-MINP, 5.6 ± 3 h for 7OH-MINP, and 5.8 ± 3.4 h for 7cx-MINP. The second elimination phase occurred approximately 24 h post dose. The mean half-life time was estimated to 5.3 ± 2 h for MINP, 6.6 ± 4 h for oxo-MINP, 5.5 ± 3 h for 7OH-MINP, and 5.8 ± 3.4 h for 7cx-MINP. During the first 24 h, 43.4% ± 24% of the dose was excreted as 34% ± 25% MINP, 3.7% ± 2% 7cx-MINP, 2.7% ± 2.3% 7OH-MINP, and 3% ± 1.6% oxo-MINP. After 24–38 h, only an additional 3.6% ± 2.5% DINP as 3% ± 2.4% MINP, 0.27% ± 0.25% oxo-MINP, 0.24% ± 0.2% 7cx-MINP, and 0.23% ± 0.3% 7OH-MINP was excreted. In conclusion, the metabolites were excreted in the following order of abundance: 7OH-MiNP (2.6% ± 1.6%) < oxo-MINP (3% ± 1.4%) < 7cx-MiNP (4% ± 2%) < MINP (37% ± 26%).

Overall, we observed a biphasic excretion for all metabolites tested (Fig. 2). A second increase occurs approximately 24 h after dose application. This effect may be explained by the fact that the metabolites as conjugates are involved in enterohepatic circulation. In addition there was an 8 h interval after 16 h and so first morning urine seems to be more highly concentrated in general. The half-life times and tmax correspond to the results observed in other studies with humans (Koch and Angerer, 2007; Koch et al., 2006, 2007; Koch et al., 2012; Kessler et al., 2012; Koch et al., 2013b). Fig. 2 shows the elimination profile of the mean DEHP-, DINP- and DINCH®-metabolites of all piglets and their corresponding dust samples. A detailed description of the toxicokinetic parameters divided by dust sample and piglet is given in the supplementary materials in Tables S4-S6. 3.1.1. Di (2-ethylhexyl) phthalate (DEHP) The mean tmax were estimated for MEHP, 5OH-MEHP, oxo-MEHP, and 5cx-MEPP as 3 ± 1 h, 2.7 ± 1 h, 3.6 ± 1.3 h and 5 ± 5 h (Fig. 2A). The half-life time of 4.2 ± 2 h for MEHP, 4.7 ± 2 h for 5OHMEHP, 6.3 ± 3.8 h for oxo-MEHP, and 5.3 ± 2.7 h for 5cx-MEPP were found in the first excretion phase. The second excretion period occurred approximately 24 h post dosing, and the mean half-life time was in decreasing order 5.6 ± 3 h for MEHP, 5.2 ± 2 h for oxo-MEHP,

3.1.3. Diisononyl 1,2-cyclohexanedicarboxylic acid (DINCH®) The mean urinary maximum concentration was reached at 10 ± 8 h (cx-MINCH), 14 ± 9 h (OH-MINCH), 16 ± 9 h (MINCH) and 19 ± 9 h (oxo-MINCH) after dosing (Fig. 2C). The mean elimination half-life time for MINCH, cx-MINCH, OH-MINCH, and oxo-MINCH in decreasing order was 15 ± 16 h, 11.5 ± 14 h, 10.5 ± 8.5 h, and

Fig. 2. Time course of the sum of the piglets mean urinary (A) DEHP-, (B) DINP-, (C) DINCH®-metabolite excretion after dust administration and their metabolites [%] in relation to the given dust dose. 86

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7 ± 5 h. During the first 24 h, 13.5% ± 18% of the given dose was eliminated as 6% ± 11% MINCH, 3.5% ± 14% oxo-MINCH, 3% ± 1.5% OH-MINCH, and 1% ± 0.7% cx-MINCH via urine. In the time period of 24–38 hours post dose, additionally only 1.6% ± 2% of the applied dose was detected as 0.8% ± 2% MINCH, 0.4% ± 0.9% oxo-MINCH, 0.3% ± 0.2% OH-MINCH, and 0.06% ± 0.08% cxMINCH. The DINCH® metabolites were excreted in the following order of abundance: cx-MINCH (1% ± 0.75%) < oxo-MINCH (1.6% ± 1%) < OH-MINCH (3.2% ± 1.6%) < MINCH (3.7% ± 2.7%).

Index (HI) was calculated. The Hazard Index (HI) is a tool for risk assessments. It is the sum of daily intake substance ). An HI < 1 will not result the hazard quotients (HQ= TDI substance in negative health effects, while an HI > 1 can pose a health risk (USEnvironmental Protection Agency, 2018). The HI was calculated to be 0.2. Thus, it can be concluded that the investigated plasticizers exposure via dust does not pose a significant health risk using the HIapproach. We are aware of the limitations in this risk assessment, that other phthalates, which are highly concentrated in dust are not included.

3.1.4. Comparison with human toxicokinetic studies We only focused on the metabolites, which were identified in previous studies by Koch et al. (2013a), Koch and Angerer (2007), Koch et al. (2006) as proper biomarkers for the phthalate and DINCH® exposure. Compared to studies performed with humans, we observed variations of the DINP and DINCH® metabolism in piglets. The DINP-metabolites are excreted in an order of abundance of OH-MINP < oxoMINP < 7cx-MINP < MINP in pigs. However, with regard to DINCH®, the carboxy metabolites were excreted with the lowest concentration and the monoester (MINCH) were excreted with the highest concentration in our study. In a DINP toxicokinetic study with human volunteers (Koch and Angerer, 2007), the following order of abundance of MINP < oxo-MINP < cx-MINP < OH-MINP was reported. For DINCH®, Koch et al. concluded that the main metabolite is OH-MINCH followed by cx-MINCH (Koch et al., 2013b). Our results indicate that piglets are slower in metabolizing DINP and DINCH® compared to humans. Another difference was identified in the DEHP metabolism. 5cxMEPP was the main excreted metabolite in our study with an order of abundance of MEHP < 5OH-MEHP < 5oxo-MEHP < 5cx-MEPP, whereas in human studies, an order of abundance of MEHP < 5oxoMEHP < 5cx-MEPP < 5OH-MEHP was reported (Koch et al., 2004). Compare to Ljungvall et al. (2004) who investigated the kinetic of DEHP and its metabolite MEHP in plasma of male piglets, our piglets reached a faster MEHP-urinary concentration maximum and their urinary MEHP - half life time was shorter than Ljunvall et al. estimated in the plasma (median: tmax: 8 h, t1/2 :6.3 h).

3.3. Dust as an exposure source Using our data, we are able to show that the bioavailability of plasticizers in dust samples collected indoors in piglets leads to a substantial intake of these substances. The background exposure in our study was kept as low as possible, verified by control urine samples and measurements of environmental media as well as food samples. Our results are in good agreement with previous in vitro digestion tests where dust was already suspected to be a relevant exposure source. In the previous study, where our dust samples originated (Fromme et al., 2013a), the phthalate metabolite concentration of 663 urine samples from children who attended the investigated daycare centers was analyzed. The daily intake was determined by back-calculation based on the amount of the urinary phthalate metabolites. A typical and high intake was calculated using the median and the 95th percentile of urinary metabolite concentration. The intake for DEHP was 3.3 μg/kg b.w. (typical) and 11.9 μg/kg b.w. (high). For DINP it was 2.2 μg/kg b.w. for the typical as well as 14.1 μg/kg b.w. for the high intake scenario. The median DINCH® intake through dust was 1 μg/kg b.w./d and the high intake was 5.4 μg/kg b.w./d (Fromme et al., 2016). Compared to the TDI value in the high scenario, 24% of the TDI for DEHP, 9% for DINP and 0.5% for DINCH® was reached (Fromme et al., 2013a, 2016). If we calculate the mean intake from dust using our measurements in dust and the obtained bioavailability, we predicted an average intake solely from dust of 11 μg/kg b.w. for DEHP, 3 μg/kg b.w. for DINP and 0.3 μg/kg b.w. for DINCH®. The mean calculated intake for DEHP is close to the high intake reported by (Fromme et al., 2013a). Because it is well-known that other factors, such as diet, have important contributions to the total DEHP intake, the amount of dust ingested per day might be lower in children than assumed. Moreover, other components of a normal mixed diet lead to a lower bioavailability in humans compared to piglets eating only potatoes. Interestingly, Heinemeyer et al. (2013) showed that the average daily dietary intake of DEHP ranged between 3–14 μg/kg b.w./d and only 30% to 40% of this is contributed by the ingestion of dust. For toddlers, the highest reported DEHP exposure through dust was 5.8 μg/kg b.w. (Wang et al., 2013). In a comprehensive study in China and the US, it was assumed that the dust contributes only 2%–5% (China) and 10%–58 % (US) of the total DEHP intake (Guo and Kannan, 2011). Additionally, (Kang et al. (2012)) estimated that a moderate dust intake of 50 mg/d contributes 28.4% to the overall DEHP exposure.

3.1.5. Limitations Additionally, it should be clarified if, e.g., 5cx-MEPP was also identified as a structural analog of 5cx-MEPTP, which is a metabolite of diethylhexyl terephthalate (DEHT), a structural isomer of DEHP (Lessmann et al., 2016). The presence of DEHT (mean 209.8 ± 85 mg/ kg) in our dust samples might lead to an unintended background exposure, which was probably measured as 5cx-MEPP of DEHP. This could lead to a minor overestimation of the bioavailability of DEHP. Because DINCH® is an isomer, our internal standard did not match perfectly with the excreted isomer of MINCH. However, we integrated the whole peak, which might lead to a minor misestimation of the excreted amount. Therefore, the results should be interpreted with caution and further verification is necessary. Although the literature indicates sex differences in the toxicokinetic, we are aware that there might be a limitation in our results because we only had male piglets. Despite those limitations, we are convinced that our data is robust enough to use for further calculations and risks assessments.

4. Conclusions Our results indicate that the bioavailability of plasticizers is less than 50%. In consideration of an estimated uptake of 50 mg of dust per day, it can be concluded that dust is in addition to diet an exposure source but, except for DEHP, still a minor contributor to the total exposure (22% TDIDEHP, 2.2% TDIDINP, and 0.3% TDIDINCH®,). Overall, dust must be considered as a phthalate exposure for toddlers. In the future, research should be focused on determining the actual amount of ingested dust, especially for children. Additionally, research should also address the bioavailability of other contaminants in dust, to determine whether dust is a source of exposure to other pollutants too.

3.2. Cumulative risk assessment With our bioavailability data and assuming a daily dust intake of 50 mg and an average bodyweight of 13 kg, the mean intake for DEHP, DINP and DINCH® through dust for a child would be 11 ± 2.5 μg/kg b.w., 3 ± 0.3 μg/kg b.w. and 0.3 ± 0.3 μg/kg b.w., respectively. This intake would contribute 22% (DEHP), 2.2% (DINP) and 0.3% (DINCH®) to the TDI of DEHP (50 μg/kg b.w./d), DINP (150 μg/kg b.w./d), and DINCH (1 mg/kg b.w./d). For a cumulative risk assessment, the hazard 87

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Declaration of Competing Interest

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