Prenatal exposure to a mixture of different phthalates increases the risk of mammary carcinogenesis in F1 female offspring.

Phthalates metabolites have been detected in the urine of pregnant and breastfeeding women. Thus, this study evaluated the adverse effects of maternal exposure to a mixture of six phthalates (Pth mix) on the mammary gland development and carcinogenesis in F1 female offspring. Pregnant female Sprague-Dawley rats were exposed daily to vehicle or Pth mix (35.22% diethyl-phthalate, 21.03% di-(2-ethylhexyl)-phthalate, 14.91% dibutyl-phthalate, 15.10% diisononyl-phthalate, 8.61% diisobutyl-phthalate, and 5.13% benzylbutyl-phthalate) by gavage at 20 µg/kg, 200 µg/kg or 200 mg/kg during gestational day 10 (GD 10) to postnatal day 21 (PND 21). After weaning (PND 22), some female offspring were euthanized for mammary gland analyses while other females received a single dose of N-methyl-N-nitrosourea (MNU, 50 mg/kg) or vehicle and then tumor incidence and multiplicity were recorded until PND 180. Maternal Pth mix exposure increased the number of Ki-67 and progesterone receptor-positive epithelial cells in the mammary gland from Pth mix 200 at µg/kg and 200 mg/kg groups. In addition, tumor incidence and mean number were higher only in Pth mix at 200 mg/kg when compared to the vehicle-treated group, and percentage of tumor-free animals was lower in Pth mix at 200 µg/kg and 200 mg/kg groups. The findings indicate that perinatal Pth mixture exposure increased susceptibility to MNU-induced mammary carcinogenesis in adult F1 female offspring.

Simultaneous determination of primary and secondary phthalate monoesters in the Taihu Lake: Exploration of sources.

While phthalates monoesters have been recognized as the bioactive metabolites of phthalates, the knowledge on their environmental occurrence and sources is limited. In this study, monomethyl phthalate (MMP), monoethyl phthalate (MEP), mono-iso-butyl phthalate (MiBP), mono-n-butyl phthalate (MnBP), and mono-2-ethylhexyl phthalate (MEHP) were frequently detected in water samples from the Taihu Lake in China using an improved SPE-LC-MS-MS method. The mean concentrations for MMP, MEP, MiBP, MnBP, and MEHP were 51.7 ±â€¯25.2, 6.0 ±â€¯4.8, 19.6 ±â€¯14.6, 42.2 ±â€¯64.7, and 33.0 ±â€¯37.4 ng/L, respectively, while those of their corresponding parent chemicals, DMP, DEP, DiBP, and DnBP and DEHP, were 36.54 ±â€¯43.22, 42.64 ±â€¯66.66, 246.8 ±â€¯311.1, 524.7 ±â€¯586.9, and 208.1 ±â€¯223.5 ng/L, respectively. Three secondary monoesters of DEHP, mono (2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono (2-ethyl-5-oxohexyl) phthalate (MEOHP), and mono (2-ethyl-5-carboxypentyl) phthalate (MECPP) were for the first time detected with mean concentration of 1.27 ±â€¯1.33, 1.33 ±â€¯1.54, and 0.73 ±â€¯0.79 ng/L, respectively. The percentage of the sum concentration of MEOHP, MEHHP, and MECPP relative to total concentration of DEHP metabolites was 5.3-12.4%. DEHP was identified to be biodegraded into secondary phthalate monoesters in water from the Taihu Lake, but their contribution to the total concentration of DEHP metabolites was 1.2-3.6%, lower than those in the Taihu Lake. Primary and secondary DEHP monoesters were also detected in influents and effluents of two sewage treatment plants adjacent to the Taihu Lake, the percentages of secondary DEHP monoesters in influents were 5.8% and 11.3%, similar with those in the Taihu Lake. Taken together with their relatively high concentrations in influents, the discharging of domestic wastewater may be an important contributor to the occurrence of phthalate monoesters in the Taihu Lake.

Gender-specific relationship between prenatal exposure to phthalates and intrauterine growth restriction.

BACKGROUND: No study has examined the association between prenatal phthalate exposure and intrauterine growth retardation (IUGR). This study aimed to investigate whether prenatal exposure to phthalates was associated with increased risk of IUGR. METHODS: A total of 126 mother-newborn pairs, including 42 IUGR cases and 84 control newborns and their mothers, were enrolled in this case-control study. Spot urine samples were collected during the third trimester of pregnancy, and 5 phthalate metabolites (mono-n-butyl phthalate (MBP), monomethyl phthalate (MMP), mono-2-ethylhexyl phthalate (MEHP), mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), and mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP)) were measured. RESULTS: Concentrations of MMP, MEHHP, MEOHP, and SumDEHP (MEHP, MEHHP, and MEOHP) were significantly higher in IUGR cases than in normal controls. In all subjects, urinary concentrations of MEHHP and MEOHP were significantly inversely associated with fetal growth indicators (birth weight and Quetelet's index). When mothers were stratified by infant sex, MEHHP and MEOHP concentrations were still negatively associated with fetal growth indicators, while no significant association was observed in females. In addition, exposure-response relationships were observed between MEHHP/SumDEHP concentrations in maternal urine and IUGR. CONCLUSION: Prenatal exposure to phthalates was associated with increased risk of IUGR, and male newborns were more sensitive to phthalates than females.

Identification and quantification of 14 phthalates and 5 non-phthalate plasticizers in PVC medical devices by GC-MS.

A GC/MS method was developed for the identification and quantification of 14 phthalates: 8 phthalates classified H360 (DBP, DEHP, BBP, DMEP, DnPP, DiPP, DPP and DiBP), 3 phthalates proposed to be forbidden in medical devices (DnOP, DiNP and DiDP) and 3 other phthalates none regulated (DMP, DCHP and DEP) which may interfere with hormone function. In order to identify and quantify other plasticizers that are commonly used in PVC medical devices such as DEHP substitute, 5 non-phthalate plasticizers (ATBC, DEHA, DEHT, TOTM, and DINCH) were included in this study. Analyses are carried out on a GC/MS system with electron impact ionization mode (EI). The separation of plasticizers is obtained on a cross-linked 5%-phenyl/95%-dimethylpolysiloxane capillary column 30m×0.25mm (i.d.)×0.25µm film thickness using a gradient temperature. Compounds quantification is performed by external calibration using an internal standard. Validation elements on standard solutions were determined using the ISO 12787 standard approach. Plasticizers are extracted from PVC medical devices using THF for dissolving the PVC part of the sample followed by precipitation of the PVC by addition of ethanol. The supernatant is injected into a GC/MS system after dilution in ethanol. Different validation elements, including extraction recoveries for all compounds or for DEHP a cross-validation of the extraction process using the European pharmacopoeia monograph 3.1.14 as reference method, are discussed. Results obtained on 61 medical devices in PVC and 12 raw materials used as plasticizers are given.

Classification of phthalates based on an in vitro neurosphere assay using rat mesencephalic neural stem cells.

Exposure to environmental neurotoxic chemicals both in utero and during the early postnatal period can cause neurodevelopmental disorders. To evaluate the disruption of neurodevelopmental programming, we previously established an in vitro neurosphere assay system using rat mesencephalic neural stem cells that can be used to evaluate. Here, we extended the assay system to examine the neurodevelopmental toxicity of the endocrine disruptors butyl benzyl phthalate, di-n-butyl phthalate, dicyclohexyl phthalate, diethyl phthalate, di(2-ethyl hexyl) phthalate, di-n-pentyl phthalate, and dihexyl phthalate at a range of concentrations (0-100 µM). All phthalates tested inhibited cell migration with a linear or non-linear range of concentrations when comparing migration distance to the logarithm of the phthalate concentrations. On the other hand, some, but not all, phthalates decreased the number of proliferating cells. Apoptotic cells were not observed upon phthalate exposure under any of the conditions tested, whereas the dopaminergic toxin rotenone induced significant apoptosis. Thus, we were able to classify phthalate toxicity based on cell migration and cell proliferation using the in vitro neurosphere assay.

Guidance on classification for reproductive toxicity under the globally harmonized system of classification and labelling of chemicals (GHS).

The Globally Harmonised System of Classification (GHS) is a framework within which the intrinsic hazards of substances may be determined and communicated. It is not a legislative instrument per se, but is enacted into national legislation with the appropriate legislative instruments. GHS covers many aspects of effects upon health and the environment, including adverse effects upon sexual function and fertility or on development. Classification for these effects is based upon observations in humans or from properly designed experiments in animals, although only the latter is covered herein. The decision to classify a substance based upon experimental data, and the category of classification ascribed, is determined by the level of evidence that is available for an adverse effect on sexual function and fertility or on development that does not arise as a secondary non-specific consequence of other toxic effect. This document offers guidance on the determination of level of concern as a measure of adversity, and the level of evidence to ascribe classification based on data from tests in laboratory animals.

Exposure to phthalates among premenstrual girls from rural and urban Gharbiah, Egypt: a pilot exposure assessment study.

BACKGROUND: Phthalates have been identified as endocrine active compounds associated with developmental and reproductive toxicity. The exposure to phthalates in premenstrual Egyptian females remains unknown. The objective of this study was to characterize phthalate exposure of a potentially vulnerable population of premenstrual girls from urban and rural Egypt. MATERIALS AND METHODS: We collected one spot urine sample from 60 10-13 year old females, 30 from rural Egypt, and 30 from urban Egypt from July to October 2009. Samples were analyzed for 11 phthalate metabolites. Additionally, we collected anthropometrics as well as questionnaire data concerning food storage behaviors, cooking practices, and cosmetic use. Phthalate metabolite concentrations were compared between urban and rural Egyptians as well as to age and gender matched Americans. RESULTS: Monoethyl phthalate (MEP), was detected at the highest concentration in urine of Egyptian girls (median: 43.2 ng/mL in rural, 98.8 ng/mL in urban). Concentrations of urinary metabolites of di-(2-ethylhexyl) phthalate and dibutyl phthalate were comparable between Egyptians and age matched US girls. Storage of food in plastic containers was a statistically significant predictor of urinary mono-isobutyl phthalate (MiBP) concentrations when comparing covariate adjusted means. CONCLUSIONS: Urinary concentrations of phthalate metabolites were similar in Egyptian and US populations, suggesting that phthalate exposure also occurs in developing nations. Dietary intake is likely an important route of exposure to phthalates in both urban and rural populations.

Finding maximal transcriptome differences between reprotoxic and non-reprotoxic phthalate responses in rat testis.

The chemical legislation of the EU, Registration, Evaluation, and Authorization of Chemicals (REACH), stipulates that about 30 000 chemical substances are to be assessed on their possible risks. Toxicological evaluation of these compounds will at least partly be based on animal testing. In particular, the assessment of reproductive toxicity is a very complicated, time-consuming and animal-demanding process. Introducing microarray-based technologies can potentially refine in vivo toxicity testing. If compounds of a distinct chemical class induce reproducible gene-expression responses with a recognizable overlap, these gene-expression signatures may indicate intrinsic features of certain compounds, including specific toxicity. In the present study, we have set out the first steps towards this approach for the reproductive toxicity of phthalates. Male rats were treated with a single dose of either reprotoxic or non-reprotoxic phthalates, and were analyzed 24 h afterwards. Subsequently, histopathological and gene-expression profiling analyses were performed. Despite ambiguous histopathological observations, we were able to identify genes with differential expression profiles between the reprotoxic phthalates and the non-reprotoxic counterparts. This shows that differences in gene-expression profiles, indicative of the type of exposure, may be detected earlier, or at lower doses, than classical pathological endpoints. These findings are promising for 'early warning' biomarker analyses and for using toxicogenomics in a category approach. Ultimately, this could lead to a more cost-effective approach for prioritizing the toxicity testing of large numbers of chemicals in a short period of time in hazard assessment of chemicals, which is one of the objectives of the REACH chemical legislation.

Detection of thyroid system-disrupting chemicals using in vitro and in vivo screening assays in Xenopus laevis.

We developed a thyroid hormone (TH) inducible primary screening assay for the identification and assessment of man-made chemicals that interfere with the TH-signalling pathway within target cells. The assay was developed in a Xenopus laevis cell line that was transduced with a self-inactivating (SIN) lentivirus vector (LV) containing a luciferase gene. The luciferase activation in this cell line was TH-specific: 3,3',5-L-triiodothyronine (T(3)) > 3,3'5-L-triiodothyroacetic acid (Triac) > 3,3',5-D-triiodothyronine (D-T(3)), > L-thyroxine (T(4)) > 3,3',5'-L-triiodothyronine (rT(3)). The application of the ligand-dependent luciferase assay for screening for thyroid system-disrupting chemicals revealed that three phthalates (dicyclohexyl phthalate, n-butylbenzyl phthalate, and di-n-butyl phthalate), two herbicides (ioxynil and pentachlorophenol) and a miticide (dicofol) had 3,3',5-L-triiodothyronine- T(3)- antagonist activity at concentrations ranging from 10(-6) to 10(-5) M. These chemicals also inhibited the expression of the endogenous primary T(3)-response TH nuclear receptor beta (TRbeta) gene. The inhibitory characteristics of these chemicals were similar for both assays performed, although the assay for T(3)-dependent activation of TRbeta gene was more sensitive than the luciferase assay. These results indicate that the luciferase assay was a rapid method with a small intra-assay variation for the primary screening of thyroid system-disrupting chemicals. Of the six chemicals, only n-butylbenzyl phthalate and pentachlorophenol exhibited T(3)-antagonist activity in an in vivo metamorphosis-based assay. It should be noted that chemicals elicited thyroid system-disrupting activity in the luciferase assay did not always interfere with the thyroid system in vivo.