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Additional file 2. Structural alerts.

In the context of the association between childhood leukemia and extremely low frequency magnetic fields (ELF-MF) exposure, the European project ARIMMORA aims to gain mechanistic insights on the impact of ELF-MF exposure in in vitro and in vivo models. In our studies the biological effects of ELF-MF exposure especially on changes in the juvenile immune system in Crl:CD1(ICR) mice were investigated. The mice were exposed (20 h/d, 7 d/wk) to continuous linear polarized sine-shaped 50 Hz electromagnetic fields of 10 mT, 1 mT, and 10 mT, starting with pregnant dams on day 10 p.c. and continuing to F1 offspring up to 90 days of age. At different time points, samples of spleen and peripheral blood were taken to evaluate potential changes in the number and activation status of T-cell, B-cell, and monocyte/macrophage compartments ex vivo by means of flow cytometry and measurement of cytokine secretion by ELISA. On day 28, a reduced number of CD8+ cytotoxic T-lymphocytes was seen in peripheral blood at all exposure levels compared to sham treatment. No alterations of this cell compartment were observed in spleen of all treatment groups. The effect was moderate but significant. Whether the observed reduction of T-lymphocytes has a functional effect cannot be definitively concluded from these studies. In addition, none of the tested doses of the ELF-MF signal mediated a significant induction of micronuclei in the peripheral blood erythrocyte fraction. The absence of an effect speaks against a profound direct DNA-damaging potential of ELF-MF exposure. The research leading to these results has received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 282891.

Exposures to extremely low frequency magnetic field (ELF-MF) may lead to disruptions of the genome and epigenome leading to childhood leukemia. Thus, in the EU-funded project ARIMMORA, we analyzed genes implicated in leukemia for aberrant transcriptomic, genetic, and epigenetic changes. These genes include cell adhesion molecule 1 (Cadm1), a tumor suppressor gene frequently inactivated in human cancer by promoter hypermethylation. We investigated by bisulfite sequencing and cloning, a 349-bp fragment encompassing 37 CpGs along the promoter and 5'-UTR regions of Cadm1 in spleen tissues of n = 10 female Crl:CD1 mice exposed for 90-days (20 h/d, 7 d/wk) starting on day 10 p.c. to 50Hz 10mT ELF-MF, and n = 10 respective shamexposed controls. CpG methylation pattern was heterogeneous within a spleen tissue, and among individual mice in both exposed and sham. Nevertheless, overall % methylation tended to be higher in 90-day exposed mice than in respective controls. Notably, we found clones exhibiting not only highly methylated CpGs but also non-CpGs, a finding reminiscent of stem cell nature or intriguing observation in the light of a recent report iPS reprogramming, and an (Baek et al. 2014)* that electromagnetic field can mediate efficient cell reprogramming into a pluripotent state. We found clones with this 'stem-cell pattern' in exposed 20/107 (19 %), and 7/94 (7 %) in sham-control mice. The non-CpG methylation was not random, but associated with highly methylated neighboring CpGs. Indeed, cytosines within two neighboring Sp1 binding sites and those cytosines within a nucleosome near the transcription start site (TSS) exhibited non-CpG methylation, consistent with the notion that the presence of non-CpG methylation ensure the stem-cell character by preventing the binding of proteins necessary for differentiation. We also detected n = 63 different sequence variations, of which 31 (49 %) in exposed mice, 19 (30 %) in control, and 13 (20 %) in both. Exposures to ELF-MF leading to reprogramming require further studies, and a single-cell or single-molecule approach would surely allow dissection of genetic and epigenetic changes resulting from ELF-MF exposures in intact tissues composed of a mixure of differentiated and stem cells, such as spleen. Determining sequence variations in clones with 'stem cell pattern' might provide additional insights into the potential risk of high-dose ELF-MF in leukemia development.

The environmental risk assessment of medicinal products for human use requires the calculation of a worst case Predicted Environmental Concentration (PEC) in surface water. This calculation is based on the assumption that the parent active ingredient is released to the wastewater after excretion from the human body. However, commonly used volatile inhalational anaesthetics are introduced into the atmosphere as the main target compart and only minor amounts (

Numerous studies on reproductive toxicity are expected to be necessary under the EU program on Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH). Therefore, it is important to analyse existing testing strategies including also the recently implemented extended one-generation reproduction toxicity study (EOGRTS, OECD guideline 443). For this purpose the responsiveness of the different generations and developmental stages in studies on reproductive toxicity is analysed and critical targets of reproductive toxicity are identified by using the Fraunhofer FeDTex database. The F1 generation is identified as most responsive generation in more than 50% of one-generation and multi-generation reproduction studies. Within the F1 generation the adult stage is mostly affected compared to the prenatal or postnatal stage. The target analysis in F1 has revealed alterations in bodyweight as highly sensitive for all developmental stages. Other important targets are the liver, kidney, testes, prostate, sperm parameters as well as developmental landmarks. The findings in the F2 generation have shown a higher responsiveness than F1 only in 3% of the studies. Although in 29 studies new effects are observed in F2 offspring compared to F1 irrespective of dose levels, overall no severe new effects have emerged that would change classification and labelling and justify an F1 mating. The presented data support the importance of F1 for risk assessment and demonstrate that the study design of the EOGRTS is a suitable alternative to two-generation studies. However, compared to a conventional one-generation study the EOGRTS may identify additional effects but will change risk assessment with respect to NOELs only in rare cases.

Numerous animal studies are required under the EU program on Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH). Developmental and reproductive toxicity studies are expected to be the major cost factor and the major consumer of animals. Thus, there is great necessity to develop new alternative testing strategies. One opportunity may be the replacement of the conventional two-generation reproduction toxicity study (OECD 416) by the recently implemented extended one generation reproduction toxicity study (EOGRTS, OECD guideline 443). Existing animal data can be integrated in the process of development by validation of alternative methods. The Fraunhofer Fertility and Developmental Toxicity in experimental animals database (FeDTex DB) currently covers 269 chemicals with 535 developmental and reproduction toxicity studies conducted in rodents and rabbits during the last three decades. Comparing the generation NOELs of multi-generation reproduction studies, the F1 generation was identified as most responsive. In contrast, the NOEL of the F2 generation was lower than the F1 NOEL only in 10% of the studies. The detailed analysis of the underlying effects in these studies left only 3 studies, where the LOEL of F2 was lower than in F1 or F0. Additionally, the observed F1-effects would clearly have triggered mating of F1 in an EOGRTS design. Thus, the impact of the F2 generation on the study NOEL is low and critical effects would not have been missed following the EOGRTS protocol. Furthermore, an impaired F1-fertility was only observed in 5 studies at the study LOEL. In all these studies, effects on reproductive organs or an impaired fertility were already adequately detected in F0. In conclusion, studies following the EOGRTS protocol may serve as an alternative in toxicity testing strategies.

The test item was a synthetic amorphous silica (SAS) coded NM-200 and available at the EU JRC nanomaterials repository. It is a nanostructured, precipitated SAS. As NM-200 is used as an ingredient in food it was the objective of this study to evaluate oral toxicity. The toxicity of NM-200 after daily oral gavage in male rats for 28 days with an additional 14-day recovery period (OECD TG 407; in addition: Si ion and Si particle analysis in selected organs) was tested. Dose levels of 100, 300 and 1000 mg/kg bw (vehicle: 0.5% methylhydroxypropylcellulose) were used. Extra groups (control recovery; high dose recovery) were kept for additional 14 days of post-observation. Body weights, food consumption, the functional observational battery (FOB) and hematology and clinical chemistry did not reveal any treatment-related effects. Also organ weights, necropsy, and histopathological examination did not show any substance-related findings in the high dose group, as compared to controls. Chemical analysis of silicon ions in blood, kidney and liver tissues did not reveal differences between the control and the high dose group. As a result, NM-200 did not cause any substance-related effects at doses up to 1000 mg/kg b.w. after oral exposure for 28 days in male Wistar (WU) rats. Therefore, the highest dose tested (1000 mg/kg b.w.) was determined to represent the NOAEL in this study. By exemplary TEM analysis no particles of NM 200 could be detected in selected tissues.

Studies on developmental and reproductive toxicity are the major cost factor and the major consumer of animals in toxicity testing. Thus, these studies are in contrast to the 3R-principle (Reduction, Refinement and Replacement of animal testing) and there is great necessity to develop new alternative in vitro and in silico methods. Existing animal data can be integrated in this process by identifying critical targets and by validation of alternative methods covering diverse adverse outcome pathways. The Fertility and Developmental Toxicity in experimental animals database (FeDTex DB) developed by Fraunhofer ITEM focusses on developmental and reproductive toxicity studies. Currently, 269 chemicals are covered with 535 studies conducted in rodents and rabbits during the last three decades. FeDTex DB is structured into three major parts: reference, study design and toxicological data. Unique tick-sheets and pick-lists were implemented for a multitude of subcategories to ensure consistent and precise database entries. The study design covers the general study data including test substance, study type, species used including strain, sex and number of animals per dose group, exposure including dosage, route of application and duration and sacrifice. Notably, a documentation of the scope of examination is also integrated in the study design to allow a matching with the toxicological effects. This is important as FeDTex DB contains studies following international guidelines as well as non-guideline studies with varying examinations. (Adverse) effects are assigned to the corresponding generation (i.e. F0, F1, F2 or F3), developmental stage (i.e. prenatal, postnatal or adult), and target/organ. Every single effect is finally characterised by a specific LOEL for the affected sex, examinations without effect result in a related NOEL. In order to determine the added value of the F2 generation in reproductive toxicity studies, NOELs of the F0, F1 and F2 generation have been compared. Further, a considerable chemical overlap with the Fraunhofer Repeated Dose Toxicity Database (RepDose DB) allows combined analysis of reproductive toxicity and repeated dose toxicity data.

Assessment of reproductive toxicity is an animal-, time- and cost-consuming process. In order to refine, replace and reduce animal testing, the use of in-silico, in-vitro and alternative in-vivo strategies is tested for their value to predict adverse reproductive outcomes. Due to the complexity of the reproductive cycle, encompassing maturation of gametes, mating, implantation of the conceptus, intra-uterine survival and maturation as well as postnatal development, no alternative testing strategies so far are able to cover all aspects of fertility. One approach should therefore be the use of existing animal study data, especially from repeated dose studies, to analyse indicators for reproductive toxicity. The current versions of OECD Guideline requirements for repeated dose toxicity include structural changes in reproductive organ tissues (mainly testes, epididymides, prostate, seminal vesicles and ovaries and uterus). Based on this information repeated dose toxicity studies are useful for selection of substances for further reproductive toxicity testing, for appropriate dose selection and for selection to incorporate additional parameters, e.g. for endocrine testing, into reproductive toxicity studies. In addition repeated dose studies can have a predictive value for detecting certain impairments of reproductive function. These conclusions have been extended by analysing animal studies from the Fraunhofer ITEM databases RepDose for repeated dose toxicity and FeDTex for reproductive toxicity. Further, based on the analysis of FeDTex, and in view of the development of AOP (adverse outcome pathway)-based approaches it should be considered to enhance the list of mandatory study parameters in repeated dose studies by hormone and sperm parameters and oestrus cyclicity in order to increase the predictivity for reproductive toxicity.