Screening for potential endocrine disruptors in fish: evidence from structural alerts and in vitro and in vivo toxicological assays.

BACKGROUND: The European chemicals' legislation REACH aims to protect man and the environment from substances of very high concern (SVHC). Chemicals like endocrine disruptors (EDs) may be subject to authorization. Identification of (potential) EDs with regard to the environment is limited because specific experimental assessments are not standard requirements under REACH. Evidence is based on a combination of in vitro and in vivo experiments (if available), expert judgement, and structural analogy with known EDs. OBJECTIVES: The objectives of this study are to review and refine structural alerts for the indication of potential estrogenic and androgenic endocrine activities based on in vitro studies; to analyze in vivo mammalian long-term reproduction studies with regard to estrogen- and androgen-sensitive endpoints in order to identify potential indicators for endocrine activity with regard to the environment; to assess the consistency of potential estrogenic and androgenic endocrine activities based on in vitro assays and in vivo mammalian long-term reproduction studies and fish life-cycle tests; and to evaluate structural alerts, in vitro assays, and in vivo mammalian long-term reproduction studies for the indication of potential estrogenic and androgenic endocrine disruptors in fish. RESULTS: Screening for potential endocrine activities in fish via estrogenic and androgenic modes of action based on structural alerts provides similar information as in vitro receptor-mediated assays. Additional evidence can be obtained from in vivo mammalian long-term reproduction studies. Conclusive confirmation is possible with fish life-cycle tests. Application of structural alerts to the more than 33,000 discrete organic compounds of the EINECS inventory indicated 3585 chemicals (approx. 11%) as potential candidates for estrogenic and androgenic effects that should be further investigated. Endocrine activities of the remaining substances cannot be excluded; however, because the structural alerts perform much better for substances with (very) high estrogenic and androgenic activities, there is reasonable probability that the most hazardous candidates have been identified. CONCLUSIONS: The combination of structural alerts, in vitro receptor-based assays, and in vivo mammalian studies may support the priority setting for further assessments of chemicals with potential environmental hazards due to estrogenic and androgenic activities.

Identification, assessment and management of "endocrine disruptors" in wildlife in the EU substance legislation--discussion paper from the German Federal Environment Agency (UBA).

A discussion paper was developed by a panel of experts of the German Federal Environment Agency (UBA) contributing to the on-going debate on the identification, assessment and management of endocrine disruptors with a view to protect wildlife according to the EU substance legislation (plant protection products, biocides, industrial chemicals). Based on a critical synthesis of the state-of-the-art regarding regulatory requirements, testing methods, assessment schemes, decision-making criteria and risk management options, we advise an appropriate and consistent implementation of this important subject into existing chemicals legislation in Europe. Our proposal for a balanced risk management of endocrine disruptors essentially advocates transparent regulatory decision making based on a scientifically robust weight of evidence approach and an adequate risk management consistent across different legislations. With respect to the latter, a more explicit consideration of the principle of proportionality of regulatory decision making and socio-economic benefits in the on-going debate is further encouraged.

The genetic origin and history of speed in the Thoroughbred racehorse.

Selective breeding for speed in the racehorse has resulted in an unusually high frequency of the C-variant (g.66493737C/T) at the myostatin gene (MSTN) in cohorts of the Thoroughbred horse population that are best suited to sprint racing. Here we show using a combination of molecular- and pedigree-based approaches in 593 horses from 22 Eurasian and North-American horse populations, museum specimens from 12 historically important Thoroughbred stallions (b.1764-1930), 330 elite-performing modern Thoroughbreds and 42 samples from three other equid species that the T-allele was ancestral and there was a single introduction of the C-allele at the foundation stages of the Thoroughbred from a British-native mare. Furthermore, we show that although the C-allele was rare among the celebrated racehorses of the 18th and 19th centuries, it has proliferated recently in the population via the stallion Nearctic (b.1954), the sire of the most influential stallion of modern time, Northern Dancer (b.1961).

Dual origins of dairy cattle farming--evidence from a comprehensive survey of European Y-chromosomal variation.

BACKGROUND: Diversity patterns of livestock species are informative to the history of agriculture and indicate uniqueness of breeds as relevant for conservation. So far, most studies on cattle have focused on mitochondrial and autosomal DNA variation. Previous studies of Y-chromosomal variation, with limited breed panels, identified two Bos taurus (taurine) haplogroups (Y1 and Y2; both composed of several haplotypes) and one Bos indicus (indicine/zebu) haplogroup (Y3), as well as a strong phylogeographic structuring of paternal lineages. METHODOLOGY AND PRINCIPAL FINDINGS: Haplogroup data were collected for 2087 animals from 138 breeds. For 111 breeds, these were resolved further by genotyping microsatellites INRA189 (10 alleles) and BM861 (2 alleles). European cattle carry exclusively taurine haplotypes, with the zebu Y-chromosomes having appreciable frequencies in Southwest Asian populations. Y1 is predominant in northern and north-western Europe, but is also observed in several Iberian breeds, as well as in Southwest Asia. A single Y1 haplotype is predominant in north-central Europe and a single Y2 haplotype in central Europe. In contrast, we found both Y1 and Y2 haplotypes in Britain, the Nordic region and Russia, with the highest Y-chromosomal diversity seen in the Iberian Peninsula. CONCLUSIONS: We propose that the homogeneous Y1 and Y2 regions reflect founder effects associated with the development and expansion of two groups of dairy cattle, the pied or red breeds from the North Sea and Baltic coasts and the spotted, yellow or brown breeds from Switzerland, respectively. The present Y1-Y2 contrast in central Europe coincides with historic, linguistic, religious and cultural boundaries.

Accurate determination of phenotypic information from historic thoroughbred horses by single base extension.

Historic DNA data have the potential to identify phenotypic information otherwise invisible in the historical, archaeological and palaeontological record. In order to determine whether a single nucleotide polymorphism typing protocol based on single based extension (SNaPshot™) could produce reliable phenotypic data from historic samples, we genotyped three coat colour markers for a sample of historic Thoroughbred horses for which both phenotypic and correct genotypic information were known from pedigree information in the General Stud Book. Experimental results were consistent with the pedigrees in all cases. Thus we demonstrate that historic DNA techniques can produce reliable phenotypic information from museum specimens.

Thresholds of toxicological concern for endocrine active substances in the aquatic environment.

The threshold of toxicological concern (TTC) concept proposes that an exposure threshold value can be derived for chemicals, below which no significant risk to human health or the environment is expected. This concept goes further than setting acceptable exposure levels for individual chemicals, because it attempts to set a de minimis value for chemicals, including those of unknown toxicity, by taking the chemical's structure or mode of action (MOA) into consideration. This study examines the use of the TTC concern concept for endocrine active substances (EAS) with an estrogenic MOA. A case study formed the basis for a workshop of regulatory, industry and academic scientists held to discuss the use of the TTC in aquatic environmental risk assessment. The feasibility and acceptability, general advantages and disadvantages, and the specific issues that need to be considered when applying the TTC concept for EAS in risk assessment were addressed. Issues surrounding the statistical approaches used to derive TTCs were also discussed. This study presents discussion points and consensus findings of the workshop.

Integrated testing and intelligent assessment-new challenges under REACH.

BACKGROUND, AIM AND SCOPE: Due to a number of drawbacks associated with the previous regime for the assessment of new and existing chemicals, the European Union established a new regulation concerning the registration, evaluation, authorisation and restriction of chemicals (REACH). All relevant industrial chemicals must now be assessed. Instead of the authorities, industry itself is responsible for the risk assessment. To achieve better and more efficient assessments while reducing animal testing, all information-standard, non-standard and non-testing-has to be used in an integrated manner. To meet these challenges, the current technical guidance documents for risk assessment of new and existing chemicals had to be updated and extended considerably. This was done by experts in a number of REACH Implementation Projects. This paper presents the most relevant results of the expert Endpoint Working Group on Aquatic Toxicity in order to illustrate the change of paradigm in the future assessment of hazards to the aquatic environment by chemical substances. MAIN FEATURES AND CHALLENGES: REACH sets certain minimum data requirements in order to achieve a high level of protection for human health and the environment. It encourages the assessor to use alternative information instead of or in addition to standard one. This information has to be equivalent to the standard information requirement and adequate to draw overall conclusions with respect to the regulatory endpoints classification and labelling, persistent, bioaccumulative and toxic (PBT) assessment and predicted no-effect concentrations (PNEC) derivation. The main task of the expert working group was to develop guidance on how to evaluate the toxicity of a substance based on integration of information from different sources and of various degrees of uncertainty in a weight of evidence approach. INTEGRATED TESTING AND INTELLIGENT ASSESSMENT: In order to verify the equivalence and adequacy of different types of information, a flexible sequence of steps was proposed, covering characterisation of the substance, analysis of modes of action, identification of possible analogues, evaluation of existing in vivo and in vitro testing data as well as of QSAR results. Finally, all available data from the different steps have to be integrated to come to an overall conclusion on the toxicity of the substance. This weight of evidence approach is the basis for the development of integrated testing strategies (ITS), in that the available evidence can help to determine subsequent testing steps and is essential for an optimal assessment. Its flexibility helps to meet the different requirements for drawing conclusions on the endpoints classification and labelling, PNEC derivation as well as PBT assessment. The integration of all kinds of additional information in a multi-criteria assessment reduces the uncertainties involved with extrapolation to the ecosystem level. The weight of evidence approach is illustrated by practical examples. CONCLUSIONS AND PERSPECTIVES: REACH leads to higher challenges in order to make sound decisions with fewer resources, i.e. to move away from extensive standard testing to an intelligent substance-tailored approach. Expert judgement and integrated thinking are key elements of the weight of evidence concept and ITS, potentially leading to better risk assessments. Important sub-lethal effects such as endocrine disruption, which are not covered by the current procedure, can be considered. Conclusions have to be fully substantiated: Risk communication will be an important aspect of future assessments.

Risk assessment of biocides in roof paint. Part 1: experimental determination and modelling of biocide leaching from roof paint.

BACKGROUND, AIM AND SCOPE: Many surface coatings, including roof paints, contain biocides. It is generally not known to what extent roof paint biocides leach from the paint, and consequently, what concentration the biocide may attain in a rainwater collection system. To this end the leaching of specific biocides from a variety of German roof paints was investigated and the resulting concentrations in collected rain water were estimated. MATERIALS AND METHODS: A laboratory simulation was used to determine the time dependant leaching rate of the biocide from the paint into synthetic rainwater. The concentrations of biocide in the leachate were quantified using HPLC. The course of the leachate concentrations over time was fitted using a simple mathematical model. This was then used to estimate concentrations of biocides in a typical household rainwater collection system over time. RESULTS: Surprisingly, the biocides found in the paints did not always concur with the declared biocides. Concerning the modelling of runoff concentrations, it was found that--under the model assumptions--the rain intensity and cumulative raining time after application are the dominant factors influencing the concentration of the biocide. At the highest modelled rain intensity of 40 mm/hour it only takes about 2 hours to reach peak concentrations lower than 0.1 mg/L, at 0.3 mm/hour it takes about 10 hours to reach peak concentrations of 1.3, 0.9, 5.2 and 1.1 mg/L for terbutryn from Emalux paint, terbutryn from Südwest paint, carbendazim from Emalux paint, and carbendazim from MIPA paint, respectively. DISCUSSION: The results confirm that biocides leached from roof paint will be present in roof runoff. The highest estimated peak concentrations are close to the water solubility of the respective biocides. This indicates that the model assumption of a concentration independent leaching rate will tendentially lead to an overestimation of the leached concentrations under these circumstances. However, under most circumstances such as higher rain intensities, and longer time after peak concentrations have been reached, the runoff concentrations are far from the solubility limit, and therefore it is proposed that the model assumptions are tenable. CONCLUSIONS: The leaching of biocides from roof paints can be roughly assessed using a relatively simple approach. The declaration of biocidal ingredients in roof paints should be improved and information on their biocide leaching behaviour should be made available. Furthermore, the estimations should be evaluated by a field study. RECOMMENDATIONS AND PERSPECTIVES: The leaching study indicated that the concentrations of selected biocides can reach significant levels, especially after low intensity rainfall. Taking into account the inherent biological activity of the substances under scrutiny, it can already be concluded that it is not advisable to use runoff water from roofs freshly painted with biocide containing roof paints. These results have been complemented by a literature search of biological effects of the investigated biocides, ecotoxicological tests with several species and a risk analysis for organisms exposed to runoff water. This will be presented in Part 2 of this contribution.

Lipophilicity parameters for ionic liquid cations and their correlation to in vitro cytotoxicity.

Regarding the great structural variability of the currently expanding group of ionic liquids, it is highly desirable to understand the basic factors affecting their toxicity in different biological systems. The present study of a set of 74 ionic liquids with imidazolium, pyrrolidinium, pyridinium, quinolinium, quaternary phosphonium and quaternary ammonium cations and the comparatively small anions Cl(-), Br(-), BF(4)(-), or PF(6)(-) demonstrates the influence of the cation lipophilicity on the cytotoxicity in IPC-81 leukemia cells from rats. The scope of this correlation is limited to ionic liquids with these or similarly small anions that are sufficiently nonreactive under physiological and chromatographic conditions and whose cation lipophilicity does not exceed a certain threshold.