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Tue Søeborg, Hanne Frederiksen and Anna-Maria Andersson

Human exposure to chemicals may be estimated by back-calculating urinary concentrations resulting from biomonitoring studies if knowledge of the chemical's toxicokinetic properties is available. In this paper, available toxicokinetic data for back-calculating urinary concentrations into daily intake values for bisphenol A (BPA), phthalates, parabens, and triclosan (TCS) are reviewed and knowledge gaps are identified. Human data is evaluated and presented with relevant animal data. Focus is on the recovery of the administered dose, the route of administration, and differences between humans and animals. Two human toxicokinetic studies are currently used to conclude that an oral dose of BPA is recoverable in urine and that no free BPA is present in plasma in spite of several contradicting biominotoring studies. Urinary recovery of an oral dose of phthalates in humans is complicated to assess due to extensive metabolism. In animals using 14C-marked phthalates, near-complete recovery is observed. An oral dose of 14C-marked parabens is also almost completely recovered in animals. In both humans and animals, however, two unspecific metabolites are formed, which complicates the back-calculation of parabens in humans. The recovery of both oral and dermal TCS in humans has been studied, but due to background levels of TCS, the back-calculation is difficult to perform. In conclusion, due to limited data, reasonable estimates of daily intake values based on urinary data are often not possible to obtain. Several knowledge gaps are identified and new studies are suggested. The route of administration used in toxicokinetic studies often does not match realistic scenarios.

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Anna-Maria Andersson, Hanne Frederiksen, Kenneth M Grigor, Jorma Toppari and Niels E Skakkebæk

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Katrine Tefre de Renzy-Martin, Hanne Frederiksen, Jeppe Schultz Christensen, Henriette Boye Kyhl, Anna-Maria Andersson, Steffen Husby, Torben Barington, Katharina M Main and Tina Kold Jensen

Many phthalates, parabens and phenols are suspected to have endocrine-disrupting properties in humans. They are found in consumer products, including food wrapping, cosmetics and building materials. The foetus is particularly vulnerable and exposure to these chemicals therefore is of concern for pregnant women. We investigated current exposure to several commonly used phthalates, parabens and phenols in healthy, pregnant Danish women. A total of 200 spot urine samples were collected between 8 and 30 weeks of gestation and analysed for metabolites of ten phenols, seven parabens and 16 phthalate by liquid chromatography–tandem mass spectrometry representing 26 non-persistent compounds. The majority of analytes were present in the urine sample collected from most women who participated. Thus, in 174 of the 200 women, metabolites of more than 13 (>50%) of 26 compounds were detected simultaneously. The number of compounds detected per woman (either as the parent compound or its metabolite(s)) ranged from 7 to 21 with a median of 16. The majority of compounds correlated positively with each other within and between chemical groups, suggesting combined exposure sources. Estimated daily intakes (DIs) of phthalates and bisphenol A (BPA) were below their individual tolerable DI (TDI) and with hazard quotients below 1. In conclusion, we found detectable levels of phthalate metabolites, parabens and phenols in almost all pregnant women, suggesting combined multiple exposures. Although the estimated DI of phthalates and BPA for an individual was below TDI, our results still raise concern, as current toxicological risk assessments in humans do not take into account simultaneous exposure. The true cumulative risk for the foetus may therefore be underestimated.

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Hanne Frederiksen, Tina Kold Jensen, Niels Jørgensen, Henriette Boye Kyhl, Steffen Husby, Niels E Skakkebæk, Katharina M Main, Anders Juul and Anna-Maria Andersson

Several non-persistent industrial chemicals have shown endocrine disrupting effects in animal studies and are suspected to be involved in human reproductive disorders. Among the non-persistent chemicals that have been discussed intensively during the past years are phthalates, bisphenol A (BPA), triclosan (TCS), and parabens because of their anti-androgenic and/or estrogenic effects. Phthalates are plasticizers used in numerous industrial products. Bisphenol A is the main component of polycarbonate plastics and epoxy resins. Parabens and TCS are antimicrobial preservatives and other phenols such as benzophenone-3 (BP-3) act as a UV-screener, while chlorophenols and phenyl phenols are used as pesticides and fungicides in agriculture. In spite of the widespread use of industrial chemicals, knowledge of exposure sources and human biomonitoring studies among different segments of the population is very limited. In Denmark, we have no survey programs for non-persistent environmental chemicals, unlike some countries such as the USA (NHANES) and Germany (GerES). However, we have analyzed the excretion of seven parabens, nine phenols, and the metabolites of eight different phthalates in urine samples collected over the past 6 years from four Danish cohorts. Here, we present biomonitoring data on more than 3600 Danish children, adolescents, young men, and pregnant women from the general population. Our study shows that nearly all Danes were exposed to the six most common phthalates, to BPA, TCS, and BP-3, and to at least two of the parabens. The exposure to other non-persistent chemicals was also widespread. Our data indicate decreasing excretion of two common phthalates (di-n-butyl phthalate and di-(2-ethylhexyl) phthalate) over time.

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Dorte Vesterholm Jensen, Jeppe Christensen, Helena E Virtanen, Niels E Skakkebæk, Katharina M Main, Jorma Toppari, Christine W Veje, Anna-Maria Andersson, Flemming Nielsen, Philippe Grandjean and Tina Kold Jensen

Geographical differences in the occurrence of diseases in male reproductive organs, including malformation in reproductive tract, between Denmark and Finland have been reported. The reason for these differences is unknown, but differences in exposure to chemicals with endocrine-disrupting abilities have been suggested. Among these chemicals are perfluoro-alkylated substances (PFASs), a group of water- and grease-repellent chemicals used in outdoor clothes, cookware, food packaging, and textiles. In this study, we, therefore, investigated differences in PFAS exposure levels between Denmark and Finland and the association between cord blood PFAS levels and congenital cryptorchidism. Boys from a joint ongoing prospective birth cohort study were included. We analyzed PFAS levels in cord blood serum samples collected from 29 Danish boys with congenital cryptorchidism, 30 healthy Danish matched controls recruited from 1997 to 2001, 30 Finnish cases, and 78 Finnish healthy matched controls recruited from 1997 to 1999. Additionally, 48 Finnish cases recruited from 2000 to 2002 were included. Perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) were detected in all the 215 Danish and Finnish cord blood samples with significantly higher levels being observed in the Danish samples (medians: PFOA, 2.6 ng/ml and PFOS, 9.1 ng/ml) than in the Finnish samples (medians: PFOA, 2.1 ng/ml and PFOS, 5.2 ng/ml). We found no associations between cord blood PFOA and PFOS levels and congenital cryptorchidism after adjustment for confounders. Our data indicate that women in Denmark and Finland are generally exposed to PFOA and PFOS but there are differences in exposure levels between countries. We found no statistically significant association between cord blood PFOA and PFOS levels and congenital cryptorchidism; however, our study was small and larger studies are warranted.

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Marie Lindhardt Johansen, Ravinder Anand-Ivell, Annette Mouritsen, Casper P Hagen, Mikkel G Mieritz, Tue Søeborg, Trine Holm Johannsen, Katharina M Main, Anna-Maria Andersson, Richard Ivell and Anders Juul

Insulin-like factor 3 (INSL3) is a promising marker of Leydig cell function with potentially high clinical relevance. Limited data of INSL3 levels in relation to other reproductive hormones in healthy pubertal boys exist. In this study, we aimed to evaluate longitudinal serum changes in INSL3 compared with LH, FSH, testosterone, inhibin B, and anti-Müllerian hormone (AMH) during puberty in healthy boys. Ten boys were included from the longitudinal part of the COPENHAGEN Puberty Study. Pubertal evaluation, including testicular volume, was performed and blood samples were drawn every 6 months for 5 years. Serum concentrations of testosterone were determined by a newly developed LC–MS/MS method, and serum concentrations of INSL3, AMH, inhibin B, FSH, and LH respectively were determined by validated immunoassays. The results showed that serum INSL3 levels increased progressively with increasing age, pubertal onset, and testicular volume. In six of the ten boys, LH increased before the first observed increase in INSL3. In the remaining four boys, the increase in LH and INSL3 was observed at the same examination. The increases in serum concentrations of LH, testosterone, and INSL3 were not parallel or in ordered succession and varied interindividually. We demonstrated that INSL3 concentrations were tightly associated with pubertal onset and increasing testicular volume. However, the pubertal increases in LH, INSL3, and testosterone concentrations were not entirely parallel, suggesting that INSL3 and testosterone may be regulated differently. Thus, we speculate that INSL3 provides additional information on Leydig cell differentiation and function during puberty compared with traditional markers of testicular function.