The OECD Validation Program of the H295R Steroidogenesis Assay for the Identification of In Vitro Inhibitors and Inducers of Testosterone and Estradiol Production. Phase 2: Inter-Laboratory Pre-Validation Studies (8 pp)

Background, Goals and Scope In response to concerns that have been raised about chemical substances that may alter the function of endocrine systems and result in adverse effects on human health, an OECD initiative was undertaken to develop and validate in vitro and in vivo assays to identify chemicals that may interfere with endocrine systems of vertebrates. Here we report on studies that were conducted to develop and standardize a cell-based screening assay using the H295R cell line to prioritize chemicals that may act on steroidogenic processes in humans and wildlife. These studies are currently ongoing as part of the ‘Special Activity on the Testing and Assessment of Endocrine Disruptors’ within the OECD Test Guidelines Program to review, develop, standardize, and validate a number of in vitro and in vivo toxicological assays for testing and assessment of chemicals concerning their potential to interact with the endocrine system of vertebrates. Study Design Six laboratories from five countries participated in the pre-validation studies. Each laboratory tested the effects of three model chemicals on the production of testosterone (T) and estradiol (E2) using the H295R Steroidogenesis Assay. Chemicals tested were well described inducers or inhibitors of steroidogenic pathways (forskolin, prochloraz and fadrozole). All experiments were conducted in 24 well plates following standard protocols. Six different doses per compound were analyzed in triplicate per plate. A quality control (QC) plate was run in conjunction with the chemical exposure plate to account for inter-assay variation. Each chemical exposure was conducted two or three times. Results All laboratories successfully detected increases and/or decreases in hormone production by H295R cells after exposure to the different model compounds and there was good agreement in the pattern of response for all groups. Forskolin increased both T and E2 while fadrozole and prochloraz decreased production of both hormones. All chemicals affected hormone production in a dose-dependent manner with the exception of fadrozole which caused maximum inhibition of E2 at the two least concentrations tested. Some inter-laboratory differences were noted in the alteration of hormone production measured in chemically exposed cells. However, with the exception of the production of T measured at one laboratory in cells exposed to forskolin, the EC₅₀s calculated were comparable (coefficients of variation 34–49%) for all hormones. Discussion and Perspectives The results indicated that the H295R Steroidogenesis Assay protocol was robust, transferable and reproducible among all laboratories. However, in several instances that were primarily related to one laboratory there were unexplained minor uncertainties related to the inter-laboratory hormone production variation. Based on the findings from this Phase 2 prevalidation study, the H295R Steroidogenesis Assay protocol is currently being refined. The next phase of the OECD validation program will test the refined protocol among the same group of laboratories using an extended set of chemicals (∼30) that will include positive and negative chemical controls as well as a broad spectrum of different potential inducers and inhibitors of steroidogenic pathways. Submission Editor: Dr. Carsten Brühl (bruehl@uni-landau.de)     

美欧环境内分泌干扰物研究框架

欧美于20世纪90年代开始系统研究环境内分泌干扰物（EDs)，为此成立了专门的工作机构负责EDs研究的规划与协调工作。研究框架包括确认EDs对人类和生态效应的方法，剂量一效应关系模型以及检测环境暴露水平等。研究对环境威胁最大受试物的主要毒理学终点有致癌性，生殖发育毒性，神经毒性和免疫毒性，目前危险评价的新进展是应用定量结构活性关系（QSAR）模型预测化学物的安全性。     

Tetrabromobisphenol A (TBBPA): A controversial environmental pollutant

Tetrabromobisphenol A (TBBPA) is one of the most widely used brominated flame retardants and is extensively used in electronic equipment, furniture, plastics, and textiles. It is frequently detected in water, soil, air, and organisms, including humans, and has raised concerns in the scientific community regarding its potential adverse health effects. Human exposure to TBBPA is mainly via diet, respiration, and skin contact. Various in vivo and in vitro studies based on animal and cell models have demonstrated that TBBPA can induce multifaceted effects in cells and animals, and potentially exert hepatic, renal, neural, cardiac, and reproductive toxicities. Nevertheless, other reports have claimed that TBBPA might be a safe chemical. In this review, we re-evaluated most of the published TBBPA toxicological assessments with the goal of reaching a conclusion about its potential toxicity. We concluded that, although low TBBPA exposure levels and rapid metabolism in humans may signify that TBBPA is a safe chemical for the general population, particular attention should be paid to the potential effects of TBBPA on early developmental stages.     

Toxics Use Reduction in the Home: Lessons Learned from Household Exposure Studies

Workers and fence-line communities have been the first to benefit from the substantial reductions in toxic chemical use and byproducts in industrial production resulting from the Massachusetts Toxics Use Reduction Act (TURA). As TURA motivates reformulation of products as well as retooling of production processes, benefits could extend more broadly to large-scale reductions in everyday exposures for the general population. Household exposure studies, including those conducted by Silent Spring Institute, show that people are exposed to complex mixtures of indoor toxics from building materials and a myriad of consumer products. Pollutants in homes are likely to have multiple health effects because many are classified as endocrine disrupting compounds (EDCs), with the ability to interfere with the body’s hormone system. Product-related EDCs measured in homes include phthalates, halogenated flame retardants, and alkylphenols. Silent Spring Institute’s chemical analysis of personal care and cleaning products confirms many are potential sources of EDCs, highlighting the need for a more comprehensive toxics use reduction (TUR) approach to reduce those exposures. Toxics use reduction targeted at EDCs in consumer products has the potential to substantially reduce occupational and residential exposures. The lessons that have emerged from household exposure research can inform improved chemicals management policies at the state and national levels, leading to safer products and widespread health and environmental benefits.     

Genotoxic effects of binary mixtures of xenoandrogens (tributyltin, triphenyltin) and a xenoestrogen (ethinylestradiol) in a partial life-cycle test with Zebrafish (Danio rerio)

A partial life-cycle test with the model fish Danio rerio was performed in order to evaluate the genotoxic potential of binary mixtures of xenoandrogenic (tributyltin--TBT; triphenyltin--TPT) and an estrogenic compound (ethinylestradiol--EE2). Five days post-fertilisation larvae were diet-exposed to environmental relevant concentrations of TBT and TPT (25 ng/g-100 ng/g), and water-exposed to ethinylestradiol (3.5 ng/L) for a four-month period; binary mixtures of TBT plus EE2 and TPT plus EE2 were run in parallel. The erythrocytic nuclear abnormalities (ENA) assay in circulating erythrocytes was used to evaluate genotoxicity in the end of the four-month exposure period. A significant increase (p<0.05, Kruskall-Wallis non-parametric ANOVA) in ENA frequency, in comparison with control animals, was observed in those animals exposed to TBT and TPT (the highest doses only), and to EE2 and the binary mixtures, although neither synergistic nor additive effects of the tested compounds were evident. Overall, the results clearly indicate that chronic exposure to low levels of TBT, TPT, EE2 and binary mixtures of TBT plus EE2 and TPT plus EE2 are genotoxic to zebrafish, which may suggest that wild fish populations may be under increased DNA damage in areas contaminated by these endocrine disrupting chemicals.     

Bisphenol A and Bisphenol S release in milk under household conditions from baby bottles marketed in Italy

A simple and sensitive validated analytical method based on liquid chromatography coupled to tandem fluorescence (FD) and ultraviolet (UV) spectrophotometry was applied to monitor the presence of bisphenol A and bisphenol S in plastic baby bottles marketed in Italy. The limits of detection (LOD) were 3.75 ng mL^?1 and 80.00 ng mL^?1, and those of quantification (LOQ) were 12.51 ng mL^?1 and 260.00 ng mL^?1 for BPA (FD detection) and for BPS (UV detection), respectively. BPA was found in only four samples, two samples undergone to microwave heating and two samples undergone to bottle warmer heating either at 40°C or at 80°C. Although the quantities of leached BPA were well below the reference dose for daily intake established by the European Food Safety Authority (EFSA) (4.0 µg kg^?1 bw/day), the release of BPA and BPS from these plastic materials should be carefully considered by the government authorities to increase people's awareness on this issue and to protect the most vulnerable population group.     

Overcoming the toxicity effects of municipal wastewater sludge and biosolid extracts in the Yeast Estrogen Screen (YES) assay

For nearly two decades, the Yeast Estrogen Screen (YES) has been used as a valuable tool for determining the total estrogenic potency of various environmental samples, including influent and effluent streams at municipal wastewater plants. However, applying the YES assay to wastewater sludges and stabilized biosolids has been problematic. This is due to co-extracted compounds from the solids either proving toxic to the yeast or masking the presence of estrogenic substances. The present research describes the development and validation of sample preparation steps that mitigate the toxicity effects of municipal wastewater sludge and biosolid samples in the YES assay, while allowing for reliable dose-dependent expression of estrogenic activity. A copper work-up for sulfur removal and chromatographic cleanup with silica and alumina were required in addition to solid-phase extraction to adequately remove interfering compounds. Sample stabilization methods such as autoclaving, lyophilization and formaldehyde treatment were found to be detrimental to the assay. Hence, heat-drying is recommended to prevent cytotoxicity and the degradation of estrogenic substances.     

Exposure assessment of French women and their newborn to brominated flame retardants: determination of tri- to deca- polybromodiphenylethers (PBDE) in maternal adipose tissue, serum, breast milk and cord serum

In the frame of a French monitoring program, tri- to deca- polybromodiphenylethers (PBDE) have been measured in maternal and cord serum, adipose tissue, and breast milk samples, collected from 93 volunteer women during caesarean deliveries. The seven major tri- to heptaBDE (BDE-28, 47, 99, 100, 153, 154, and 183) were detected in adipose tissue and breast milk with cumulated median values of 2.59 and 2.51 ng g⁻¹ l w. Nine highly brominated octa- to decaBDE (BDE-196, 197, 201, 202, 203, 206, 207, 208 and 209) was performed in the same samples, with cumulated median values of 2.73 and 3.39 ng g⁻¹ l w in adipose tissue and breast milk, respectively. At this opposite, median levels of octa- to decaBDE in maternal and cord serum appeared significantly higher than the levels of tri- to heptaBDE in the same matrices, i.e. 8.85 and 12.34 versus 0.98 and 0.69 ng g⁻¹ l w, respectively.     

Mass fluxes and spatial trends of xenobiotics in the waters of the city of Halle, Germany

The behaviour and the effects of xenobiotics including pharmaceuticals and fragrances in the environment are widely unknown. In order to improve our knowledge, field investigations and modelling approaches for the entire area of the city of Halle/Saale, Germany, were performed. The distribution of the concentration values and mass fluxes are exemplified using indicators such as Bisphenol A, t-Nonylphenol, Carbamacepine, Galaxolide, Tonalide, Gadolinium and isotopes. Concentrations at a magnitude of ng/L to microg/L were found ubiquitously in the ground and surface waters. Using the concentration values, the impact of the city concerning the indicators was not always evident. Only the assessment of the mass fluxes shows significant urban impacts along the city passage. The calculation of the mass fluxes shows increasing values for all investigated xenobiotics during the city passage; only Bisphenol A stagnates. A balance model of water and indicator mass fluxes was built up for the entire city area.     

Occurrence and temporal variations of the xenoestrogens bisphenol A, 4-<Emphasis Type="Italic">tert</Emphasis>-octylphenol,and tech. 4-nonylphenol in two German wastewater treatment plants

GOAL, SCOPE, AND BACKGROUND: The xenoestrogens bisphenol A, 4-tert-octylphenol, and the technical isomer mixture of 4-nonylphenol (tech. 4-nonylphenol) belong to the group of chemicals which are called endocrine disrupters due to their property of causing hormonal dysfunctions in the endocrine system of organisms at very low concentrations. Bisphenol A, 4-tert-octylphenol, and the tech. 4-nonylphenol (mixture of isomers) were determined in water samples collected from the influent and effluent of two German wastewater treatment plants (WWTP) during a long-time sampling period from February 2003 till August 2005 to assess their occurrence and temporal variations in WWTPs. METHODS: The compounds were extracted and concentrated from water by solid-phase extraction (SPE) using Bond Elut PPL cartridges and quantified by use of gas chromatography-mass spectrometry (GC-MS). RESULTS: The influent concentrations were as follows: Bisphenol A < limit of detection of the method (< ldm)--12,205 ng L(-1), tech. 4-nonylphenol < ldm--10,186 ng L(-1), and 4-tert-octylphenol 39-1,495 ng L(-1). The measured effluent concentrations were lower with values in the range of < ldm--7,625 ng L(-1) for bisphenol A, < ldm--14,444 ng L(-1) for tech. 4-nonylphenol, and < ldm--392 ng L(-1) for 4-tert-octylphenol. All target compounds were largely eliminated during the wastewater treatment process. The elimination efficiency varied between 73% and 93%. DISCUSSION: All analytes show highly fluctuating influent concentrations with very high peak concentrations at particular sampling times. The variation of effluent concentrations is by far lower than the variation of influent concentrations. For tech. 4-nonylphenol, a significant temporal concentration variation has been detected with very high concentrations up to the microgram-per-liter level in the time from February 2003 till July 2003 and clearly decreasing concentrations in the time from June 2004 till August 2005. This corresponds well with the implementation of Directive 2003/53/EC (nonylphenol and nonylphenol ethoxylates in the European Union "may not be placed on the marked or used as a substance or constituent of preparations in concentrations equal or higher than 0.1% by mass") from January 2005 on. Bisphenol A is present in the effluent samples in a wide range of concentrations from below the detection limit to high concentrations up to the microgram-per-liter level. For 4-tert-octylphenol, no particular trend of concentration development has been observed. CONCLUSIONS: Combined SPE and GC-MS proved to be an efficient method to identify and quantify polar organic compounds in environmental samples. With respect to the concentrations measured in the present study, bisphenol A sometimes is the prominent compound in influent samples. Neither bisphenol A nor 4-tert-octylphenol or tech. 4-nonylphenol show seasonal variations. However, there was a significant general trend of decreasing concentrations of tech. 4-nonylphenol in influent and effluent samples from both WWTPs which probably reflects the implementing Directive 2003/53/EC. RECOMMENDATIONS AND PERSPECTIVES: Further research is needed to investigate whether the observed decrease of tech. 4-nonylphenol concentrations in German WWTPs since June 2004 will continue further on. The reason for the high effluent concentrations of bisphenol A in only a few samples has to be clarified in further research. The results from this study provide insight into the concentration development of the xenoestrogens bisphenol A, tech. 4-nonylphenol, and 4-tert-octylphenol in WWTPs in the time span between 2003 and 2005.