Assessing the Influence of Propylthiouracil and Phenytoin on the Metabolomes of the Thyroid, Liver, and Plasma in Rats.

The thyroid hormones (THs) regulate various physiological mechanisms in mammals, such as cellular metabolism, cell structure, and membrane transport. The therapeutic drugs propylthiouracil (PTU) and phenytoin are known to induce hypothyroidism and decrease blood thyroid hormone levels. To analyze the impact of these two drugs on systemic metabolism, we focused on metabolic changes after treatment. Therefore, in a rat model, the metabolome of thyroid and liver tissue as well as from the blood plasma, after 2-week and 4-week administration of the drugs and after a following 2-week recovery phase, was investigated using targeted LC-MS/MS and GC-MS. Both drugs were tested at a low dose and a high dose. We observed decreases in THs plasma levels, and higher doses of the drugs were associated with a high decrease in TH levels. PTU administration had a more pronounced effect on TH levels than phenytoin. Both drugs had little or no influence on the metabolomes at low doses. Only PTU exhibited apparent metabolome alterations at high doses, especially concerning lipids. In plasma, acylcarnitines and triglycerides were detected at decreased levels than in the controls after 2- and 4-week exposure to the drug, while sphingomyelins and phosphatidylcholines were observed at increased levels. Interestingly, in the thyroid tissue, triglycerides were observed at increased concentrations in the 2-week exposure group to PTU, which was not observed in the 4-week exposure group and in the 4-week exposure group followed by the 2-week recovery group, suggesting an adaptation by the thyroid tissue. In the liver, no metabolites were found to have significantly changed. After the recovery phase, the thyroid, liver, and plasma metabolomic profiles showed little or no differences from the controls. In conclusion, although there were significant changes observed in several plasma metabolites in PTU/Phenytoin exposure groups, this study found that only PTU exposure led to adaptation-dependent changes in thyroid metabolites but did not affect hepatic metabolites.

Gut microbiome and plasma metabolome changes in rats after oral gavage of nanoparticles: sensitive indicators of possible adverse health effects.

BACKGROUND: The oral uptake of nanoparticles is an important route of human exposure and requires solid models for hazard assessment. While the systemic availability is generally low, ingestion may not only affect gastrointestinal tissues but also intestinal microbes. The gut microbiota contributes essentially to human health, whereas gut microbial dysbiosis is known to promote several intestinal and extra-intestinal diseases. Gut microbiota-derived metabolites, which are found in the blood stream, serve as key molecular mediators of host metabolism and immunity. RESULTS: Gut microbiota and the plasma metabolome were analyzed in male Wistar rats receiving either SiO2 (1000 mg/kg body weight/day) or Ag nanoparticles (100 mg/kg body weight/day) during a 28-day oral gavage study. Comprehensive clinical, histopathological and hematological examinations showed no signs of nanoparticle-induced toxicity. In contrast, the gut microbiota was affected by both nanoparticles, with significant alterations at all analyzed taxonomical levels. Treatments with each of the nanoparticles led to an increased abundance of Prevotellaceae, a family with gut species known to be correlated with intestinal inflammation. Only in Ag nanoparticle-exposed animals, Akkermansia, a genus known for its protective impact on the intestinal barrier was depleted to hardly detectable levels. In SiO2 nanoparticles-treated animals, several genera were significantly reduced, including probiotics such as Enterococcus. From the analysis of 231 plasma metabolites, we found 18 metabolites to be significantly altered in Ag-or SiO2 nanoparticles-treated rats. For most of these metabolites, an association with gut microbiota has been reported previously. Strikingly, both nanoparticle-treatments led to a significant reduction of gut microbiota-derived indole-3-acetic acid in plasma. This ligand of the arylhydrocarbon receptor is critical for regulating immunity, stem cell maintenance, cellular differentiation and xenobiotic-metabolizing enzymes. CONCLUSIONS: The combined profiling of intestinal microbiome and plasma metabolome may serve as an early and sensitive indicator of gut microbiome changes induced by orally administered nanoparticles; this will help to recognize potential adverse effects of these changes to the host.

ChemBioSim: Enhancing Conformal Prediction of In Vivo Toxicity by Use of Predicted Bioactivities.

Computational methods such as machine learning approaches have a strong track record of success in predicting the outcomes of in vitro assays. In contrast, their ability to predict in vivo endpoints is more limited due to the high number of parameters and processes that may influence the outcome. Recent studies have shown that the combination of chemical and biological data can yield better models for in vivo endpoints. The ChemBioSim approach presented in this work aims to enhance the performance of conformal prediction models for in vivo endpoints by combining chemical information with (predicted) bioactivity assay outcomes. Three in vivo toxicological endpoints, capturing genotoxic (MNT), hepatic (DILI), and cardiological (DICC) issues, were selected for this study due to their high relevance for the registration and authorization of new compounds. Since the sparsity of available biological assay data is challenging for predictive modeling, predicted bioactivity descriptors were introduced instead. Thus, a machine learning model for each of the 373 collected biological assays was trained and applied on the compounds of the in vivo toxicity data sets. Besides the chemical descriptors (molecular fingerprints and physicochemical properties), these predicted bioactivities served as descriptors for the models of the three in vivo endpoints. For this study, a workflow based on a conformal prediction framework (a method for confidence estimation) built on random forest models was developed. Furthermore, the most relevant chemical and bioactivity descriptors for each in vivo endpoint were preselected with lasso models. The incorporation of bioactivity descriptors increased the mean F1 scores of the MNT model from 0.61 to 0.70 and for the DICC model from 0.72 to 0.82 while the mean efficiencies increased by roughly 0.10 for both endpoints. In contrast, for the DILI endpoint, no significant improvement in model performance was observed. Besides pure performance improvements, an analysis of the most important bioactivity features allowed detection of novel and less intuitive relationships between the predicted biological assay outcomes used as descriptors and the in vivo endpoints. This study presents how the prediction of in vivo toxicity endpoints can be improved by the incorporation of biological information-which is not necessarily captured by chemical descriptors-in an automated workflow without the need for adding experimental workload for the generation of bioactivity descriptors as predicted outcomes of bioactivity assays were utilized. All bioactivity CP models for deriving the predicted bioactivities, as well as the in vivo toxicity CP models, can be freely downloaded from https://doi.org/10.5281/zenodo.4761225.

Safety evaluation of a ß-mannanase enzyme preparation produced with Thermothelomyces thermophilus expressing a protein-engineered ß-mannanase gene.

Mannanase 19287 enzyme is an engineered ß-mannanase that can be added to diets for animals raised for human consumption to hydrolyze ß-mannans. Established toxicological analyses were conducted with the enzyme preparation to ensure the safety of this product for the intended use. The mannanase 19287 preparation was produced with Thermothelomyces thermophilus strain DSM 33149. In vitro toxicity studies presented here used dosages of the mannanase 19287 test articles up to 5000 µg/plate. For in vivo toxicity studies in Wistar rats, test articles were administered at 5.1 mg/L for inhalation toxicity and up to 15,000 mg/kg rat feed for oral toxicity, based on the Total Organic Solids (TOS) content in each test article. No treatment related adverse effects were reported in any study. The No Observed Adverse Effect Levels in the high dose group of the subchronic oral toxicity study were calculated as 1117-1298 mg TOS/kg bw/day in rats. Comparing these values to an Estimated Daily Intake for poultry demonstrated safety factors larger than 5000. Our results confirm that T. thermophilus fulfills the recognized safety criteria for the manufacture of food enzyme preparations and represent the first peer-reviewed safety evaluation of an enzyme preparation by T. thermophilus. The results of the toxicity studies presented herein attest to the safety of the mannanase 19287 enzyme for its intended use.

Prospects and challenges of multi-omics data integration in toxicology.

Exposure of cells or organisms to chemicals can trigger a series of effects at the regulatory pathway level, which involve changes of levels, interactions, and feedback loops of biomolecules of different types. A single-omics technique, e.g., transcriptomics, will detect biomolecules of one type and thus can only capture changes in a small subset of the biological cascade. Therefore, although applying single-omics analyses can lead to the identification of biomarkers for certain exposures, they cannot provide a systemic understanding of toxicity pathways or adverse outcome pathways. Integration of multiple omics data sets promises a substantial improvement in detecting this pathway response to a toxicant, by an increase of information as such and especially by a systemic understanding. Here, we report the findings of a thorough evaluation of the prospects and challenges of multi-omics data integration in toxicological research. We review the availability of such data, discuss options for experimental design, evaluate methods for integration and analysis of multi-omics data, discuss best practices, and identify knowledge gaps. Re-analyzing published data, we demonstrate that multi-omics data integration can considerably improve the confidence in detecting a pathway response. Finally, we argue that more data need to be generated from studies with a multi-omics-focused design, to define which omics layers contribute most to the identification of a pathway response to a toxicant.

KnowTox: pipeline and case study for confident prediction of potential toxic effects of compounds in early phases of development.

Risk assessment of newly synthesised chemicals is a prerequisite for regulatory approval. In this context, in silico methods have great potential to reduce time, cost, and ultimately animal testing as they make use of the ever-growing amount of available toxicity data. Here, KnowTox is presented, a novel pipeline that combines three different in silico toxicology approaches to allow for confident prediction of potentially toxic effects of query compounds, i.e. machine learning models for 88 endpoints, alerts for 919 toxic substructures, and computational support for read-across. It is mainly based on the ToxCast dataset, containing after preprocessing a sparse matrix of 7912 compounds tested against 985 endpoints. When applying machine learning models, applicability and reliability of predictions for new chemicals are of utmost importance. Therefore, first, the conformal prediction technique was deployed, comprising an additional calibration step and per definition creating internally valid predictors at a given significance level. Second, to further improve validity and information efficiency, two adaptations are suggested, exemplified at the androgen receptor antagonism endpoint. An absolute increase in validity of 23% on the in-house dataset of 534 compounds could be achieved by introducing KNNRegressor normalisation. This increase in validity comes at the cost of efficiency, which could again be improved by 20% for the initial ToxCast model by balancing the dataset during model training. Finally, the value of the developed pipeline for risk assessment is discussed using two in-house triazole molecules. Compared to a single toxicity prediction method, complementing the outputs of different approaches can have a higher impact on guiding toxicity testing and de-selecting most likely harmful development-candidate compounds early in the development process.

Metazachlor: Mode of action analysis for rat liver tumour formation and human relevance.

In a 2-year study the herbicide metazachlor (BAS 479H) was shown to significantly increase the incidence of liver tumours in female Wistar rats at a dietary level of 8000 ppm. As metazachlor is not a genotoxic agent, a series of in vivo and in vitro investigative studies were undertaken to elucidate the mode of action (MOA) for metazachlor-induced female rat liver tumour formation. Male and female Wistar rats were given diets containing 0 (control), 200 and 8000 ppm metazachlor for 3, 7, 14 and 28 days. The treatment of male rats with 200 and 8000 ppm metazachlor and female rats with 8000 ppm metazachlor resulted in significant increases in relative liver weight, which was associated with a centrilobular hepatocyte hypertrophy. Hepatocyte replicative DNA synthesis (RDS) was significantly increased in male rats given 8000 ppm metazachlor for 3 and 7 days and in female rats given 200 ppm metazachlor for 7-28 days and 8000 ppm metazachlor for 3-28 days. Significant increases in relative liver weight, centrilobular hepatocyte hypertrophy and hepatocyte RDS were also observed in male and female Wistar rats given and 500 ppm sodium phenobarbital (NaPB) for 3-28 days. The treatment of female Wistar rats with either 8000 ppm metazachlor for 7 days or with 500 ppm NaPB for 3 and 7 days resulted in the nuclear translocation of the hepatic constitutive androstane receptor (CAR). Treatment of male and female Wistar rats with 8000 ppm metazachlor for 14 days resulted in significant increases in hepatic microsomal total cytochrome P450 (CYP) content, CYP2B subfamily-dependent enzyme activities and mRNA levels, together with some increases in CYP3A enzyme activity and mRNA levels. The treatment of male Wistar rat hepatocytes with metazachlor (concentration range 0.5-50 µM) and NaPB (500 µM) for 4 days resulted in increased CYP2B enzyme activities and mRNA levels; with metazachlor and NaPB also producing significant increases in hepatocyte RDS levels. Studies were also performed with hepatocytes from male Sprague-Dawley wild type (WT) rats and CAR knockout (CAR KO) rats. While both treatment with metazachlor and NaPB for 4 days increased CYP2B enzyme activities and mRNA levels in WT rat hepatocytes, only minor effects were observed in CAR KO rat hepatocytes. Treatment with both metazachlor and NaPB only increased RDS in WT but not in CAR KO rat hepatocytes. The treatment of hepatocytes from two male human donors with 0.5-25 µM metazachlor or 500 µM NaPB for 4 days resulted in increases in CYP2B6 and CYP3A4 mRNA levels but had no effect on hepatocyte RDS. EGF as concurrently used positive control demonstrated the expected RDS response in all rat and human hepatocyte cultures. In conclusion, a series of in vivo and in vitro investigative studies have demonstrated that metazachlor is a CAR activator in rat liver, with similar properties to the prototypical CAR activator phenobarbital. A robust MOA for metazachlor-induced female rat liver tumour formation has been established. Based on the lack of effect of metazachlor on RDS in human hepatocytes, it is considered that the MOA for metazachlor-induced rat liver tumour formation is qualitatively not plausible for humans.

Innovative selection approach for a new antifungal agent mefentrifluconazole (Revysol®) and the impact upon its toxicity profile.

Mefentrifluconazole (trade name: Revysol®) is an agrochemical active ingredient from the new sub-class of isopropanol-triazole fungicides, with high selective fungicide activity. A full program of toxicity testing conducted according to OECD guidelines has shown mefentrifluconazole (MFZ) to be non-genotoxic and non-carcinogenic. Repeated dose studies in rats, mice and dogs identified the liver as the main target organ. Prenatal developmental toxicity studies in rats and rabbits did not indicate treatment-related embryofetal toxicity or teratogenicity up to the highest dose levels tested. In a two-generation dietary study in rats, the high dose level resulted in reduced food consumption and body weight gain throughout the dosing-period. Mating performance and fertility, estrous cycles, gestation length and pre-and post-natal survival of offspring were essentially unaffected and there was no evidence of masculinization of female pups or feminization of male pups. The screening strategy that led to the selection of MFZ was aimed to identify candidates with both high fungicidal activity and minimal likelihood of adverse side effects thought to arise from aromatase inhibition. The success of the selection strategy has been illustrated for MFZ by the absence in toxicity studies of effects that would indicate an endocrine disrupting potential.

Safety assessment of EPA+DHA canola oil by fatty acid profile comparison to various edible oils and fat-containing foods and a 28-day repeated dose toxicity study in rats.

The omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are recognized for their health-promoting qualities. Marine fish and fish oil currently provide the main sources of EPA and DHA for human consumption. An alternative plant-based source of EPA and DHA is provided by EPA + DHA canola event LBFLFK (LBFLFK). A comparative analysis and a 28-day toxicity study assessed the safety of LBFLFK refined, bleached, and deodorized (RBD) oil. Thirty-one different commercially-obtained fat and oil samples were tested, and principal component analysis showed that the overall fatty acid profile of LBFLFK RBD oil was most similar to Mortierella alpina oil and salmon flesh. Samples with the fewest differences in the presence or absence of individual fatty acids compared to LBFLFK RBD oil were menhaden oil and some other fish oils. In a 28-day toxicity study, LBFLFK RBD oil was administered by oral gavage to male and female Wistar rats. No signs of toxicity were evident and no adverse effects were noted in clinical observations, clinical pathology, or histopathology. Overall, these studies support the safety of LBFLFK RBD oil as a source of EPA and DHA for human consumption.

Defining baseline epigenetic landscapes in the rat liver.

AIM: Characterization of the hepatic epigenome following exposure to chemicals and therapeutic drugs provides novel insights into toxicological and pharmacological mechanisms, however appreciation of genome-wide inter- and intra-strain baseline epigenetic variation, particularly in under-characterized species such as the rat is limited. Material & methods: To enhance the utility of epigenomic endpoints safety assessment, we map both DNA modifications (5-methyl-cytosine and 5-hydroxymethyl-cytosine) and enhancer related chromatin marks (H3K4me1 and H3K27ac) across multiple male and female rat livers for two important outbred laboratory rat strains (Sprague-Dawley and Wistar). Results & conclusion: Integration of DNA modification, enhancer chromatin marks and gene expression profiles reveals clear gender-specific chromatin states at genes which exhibit gender-specific transcription. Taken together this work provides a valuable baseline liver epigenome resource for rat strains that are commonly used in chemical and pharmaceutical safety assessment.

Framework for the quantitative weight-of-evidence analysis of 'omics data for regulatory purposes.

A framework for the quantitative weight-of-evidence (QWoE) analysis of 'omics data for regulatory purposes is presented. The QWoE framework encompasses seven steps to evaluate 'omics data (also together with non-'omics data): (1) Hypothesis formulation, identification and weighting of lines of evidence (LoEs). LoEs conjoin different (types of) studies that are used to critically test the hypothesis. As an essential component of the QWoE framework, step 1 includes the development of templates for scoring sheets that predefine scoring criteria with scores of 0-4 to enable a quantitative determination of study quality and data relevance; (2) literature searches and categorisation of studies into the pre-defined LoEs; (3) and (4) quantitative assessment of study quality and data relevance using the respective pre-defined scoring sheets for each study; (5) evaluation of LoE-specific strength of evidence based upon the study quality and study relevance scores of the studies conjoined in the respective LoE; (6) integration of the strength of evidence from the individual LoEs to determine the overall strength of evidence; (7) characterisation of uncertainties and conclusion on the QWoE. To put the QWoE framework in practice, case studies are recommended to confirm the relevance of its different steps, or to adapt them as necessary.

Applying 'omics technologies in chemicals risk assessment: Report of an ECETOC workshop.

Prevailing knowledge gaps in linking specific molecular changes to apical outcomes and methodological uncertainties in the generation, storage, processing, and interpretation of 'omics data limit the application of 'omics technologies in regulatory toxicology. Against this background, the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) convened a workshop Applying 'omics technologies in chemicals risk assessment that is reported herein. Ahead of the workshop, multi-expert teams drafted frameworks on best practices for (i) a Good-Laboratory Practice-like context for collecting, storing and curating 'omics data; (ii) the processing of 'omics data; and (iii) weight-of-evidence approaches for integrating 'omics data. The workshop participants confirmed the relevance of these Frameworks to facilitate the regulatory applicability and use of 'omics data, and the workshop discussions provided input for their further elaboration. Additionally, the key objective (iv) to establish approaches to connect 'omics perturbations to phenotypic alterations was addressed. Generally, it was considered promising to strive to link gene expression changes and pathway perturbations to the phenotype by mapping them to specific adverse outcome pathways. While further work is necessary before gene expression changes can be used to establish safe levels of substance exposure, the ECETOC workshop provided important incentives towards achieving this goal.

Investigations on the dose-response relationship of combined exposure to low doses of three anti-androgens in Wistar rats.

The current investigation examines whether combined exposure to three anti-androgens (flutamide, prochloraz, vinclozolin) result in interference with endocrine homeostasis when applied at very low dose levels, and whether the results of combined exposure are more pronounced than to the individual compounds. A pre-post-natal in vivo study design was chosen with more parameters than regulatory testing protocols require (additional endpoints addressing hormone levels, morphology and histopathological examinations). Dose levels were chosen to represent the lowest observed adverse effect level (LOAEL), the no observed adverse effect level (NOAEL), and the acceptable daily intake for each individual substance. Anti-androgenic changes were observable at the effect level (LOAEL) but not at lower exposures. Nipple/areola counts appeared to be a sensitive measure of effect, in addition to male sex organ weights at sexual maturation, and finally gross findings. The results indicate the absence of evidence for effects at low or very low dose levels. No (adverse) effects were seen at the NOAEL dose. A non-monotonic dose-response relationship was not evident. Combined exposure at LOAEL level resulted in enhanced responses for anogenital index, number of areolas/nipples, delayed preputial separation and reduced ventral prostate weight in comparison to the individual compounds.

Comparing effect levels of regulatory studies with endpoints derived in targeted anti-androgenic studies: example prochloraz.

Prochloraz is an imidazole fungicide, and its regulatory toxicological data package has been primarily generated in the 1990s. More recently, studies have been published demonstrating an interaction with hormone receptors/steroidogenesis and effects with an endocrine mode of action. In the present study, prochloraz has been investigated in a comprehensive in vivo study including relevant elements of current regulatory reproduction toxicity studies and additional mechanistic parameters. Prochloraz was administered per gavage in oil from GD 6 to PND 83 to pregnant and lactating Wistar rats and their respective offspring, at doses of 0.01 mg/kg bw/day (acceptable daily intake of prochloraz), 5 mg/kg bw/day [expected no-observed-effect-level (NOEL)] and 30 mg/kg bw/day. At 30 mg/kg bw/day maternal and offspring effects (decreased viability, lower number of live offspring) were seen including a delayed entry into male puberty (+1 day) accompanied by lower male offspring body weights, increased anogenital distance/index in females and transiently retained nipples in males at PND 12 (not seen at PND 20). The only finding at the "expected NOEL" was increased incidences of transiently retained nipples in males which are not considered adverse. No effects were seen in the low-dose group. There was no evidence for a non-monotonic dose-response curve or effects at low levels.

Investigations of putative reproductive toxicity of low-dose exposures to vinclozolin in Wistar rats.

The current investigation examines whether the fungicide vinclozolin, which has an anti-androgenic mode of action, is capable of disrupting endocrine homeostasis at very low doses. The data generated clarify whether a non-monotonic dose-response relationship exists to enhance the current debate about the regulation of endocrine disruptors. Moreover, it is part of a series of investigations assessing the dose-response relationship of single and combined administration of anti-androgenic substances. A pre-postnatal in vivo study design was chosen which was compliant with regulatory testing protocols. The test design was improved by additional endpoints addressing hormone levels, morphology and histopathological examinations. Doses were chosen to represent an effect level (20 mg/kg bw/d), the current NOAEL (4 mg/kg bw/d), and a dose close to the "ADI" (0.005 mg/kg bw/d) for the detection of a possible non-monotonic dose-response curve. Anti-androgenic changes were observable at the effect level but not at lower exposures. Nipple/areola counts appeared to be the most sensitive measure of effect, followed by male sex organ weights at sexual maturation, and finally gross and histopathological findings. The results indicate the absence of evidence for effects at low or very low dose levels. A non-monotonic dose-response relationship was not evident.

Acute oral toxicity testing: Scientific evidence and practicability should govern Three Rs activities.

Acute oral toxicity is determined for regulatory hazard classification or non-classification. The European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) recommends the following modules for acute oral toxicity testing: a) the use of the in vitro 3T3 Neutral Red Uptake (NRU) test to identify substances not requiring classification and to estimate starting doses for in vivo acute oral toxicity studies; and b) the use of data from sub-acute toxicity studies to identify substances not requiring classification. However, the application of these modules in a regulatory context would require a predefined, validated and formally accepted testing strategy and data interpretation procedure, which are not available. Furthermore, the application of the 3T3 NRU assay for starting dose estimations could in fact increase the number of animals used. Finally, only very few substances exist for which data from sub-acute or other repeated dose studies are available, but data from acute studies are not. Therefore, in practice, the prediction of acute toxicity by using sub-acute toxicity data is generally irrelevant. It could even lead to a risk of overdosing in the range-finding study, which may result in the death of many or all of the animals used.

Advancing the use of noncoding RNA in regulatory toxicology: Report of an ECETOC workshop.

The European Centre for the Ecotoxicology and Toxicology of Chemicals (ECETOC) organised a workshop to discuss the state-of-the-art research on noncoding RNAs (ncRNAs) as biomarkers in regulatory toxicology and as analytical and therapeutic agents. There was agreement that ncRNA expression profiling data requires careful evaluation to determine the utility of specific ncRNAs as biomarkers. To advance the use of ncRNA in regulatory toxicology, the following research priorities were identified: (1) Conduct comprehensive literature reviews to identify possibly suitable ncRNAs and areas of toxicology where ncRNA expression profiling could address prevailing scientific deficiencies. (2) Develop consensus on how to conduct ncRNA expression profiling in a toxicological context. (3) Conduct experimental projects, including, e.g., rat (90-day) oral toxicity studies, to evaluate the toxicological relevance of the expression profiles of selected ncRNAs. Thereby, physiological ncRNA expression profiles should be established, including the biological variability of healthy individuals. To substantiate the relevance of key ncRNAs for cell homeostasis or pathogenesis, molecular events should be dose-dependently linked with substance-induced apical effects. Applying a holistic approach, knowledge on ncRNAs, 'omics and epigenetics technologies should be integrated into adverse outcome pathways to improve the understanding of the functional roles of ncRNAs within a regulatory context.

Postnatal fate of prenatal-induced fetal alterations in laboratory animals.

Currently it is common practice to evaluate the developmental toxicity hazard of chemicals or pharmaceuticals by evaluation of fetuses after administration of the compound to pregnant animals. These studies are designed to provide possible compound-related fetal changes near term, which are usually classified into malformations or variations. Malformations, but not variations are expected to adversely affect the survival or health. Therefore, classification has striking different regulatory consequences. For categorization as variation reversibility is an important criterion, but it is usually not examined in a standard guideline study. Although this issue has already been recognized long time ago, data dealing with the postnatal reversibility of fetal alterations are still rare. In the current review, literature data, regulatory documents as well as in-house data were compiled. Beside skeletal alterations of skull, vertebral column, ribs, shoulder and pelvic girdle, and extremities, kidney and heart defects are discussed and assessed.

Investigations of putative reproductive toxicity of low-dose exposures to flutamide in Wistar rats.

The current investigation examines whether the model anti-androgenic substance flutamide is capable of disrupting endocrine homeostasis at very low doses. The data generated clarify whether a non-monotonic dose-response relationship exists to enhance the current debate about the regulation of endocrine disruptors. Moreover, it is part of a series of investigations assessing the dose-response relationship of single and combined administration of anti-androgenic substances. A pre-postnatal in vivo study design was chosen, which was compliant with regulatory testing protocols. The test design was improved by additional endpoints addressing hormone levels, morphology, and histopathological examinations. Doses were chosen to represent a clear effect level (2.5 mg/kg bw/d), a low endocrine effect level (LOAEL, 0.25 mg/kg bw/d), a NOAEL for endocrine effects (0.025 mg/kg bw/d), a further dose at 0.0025 mg/kg bw/d flutamide, as well as an "ADI" (0.00025 mg/kg bw/d or 100-fold below the NOAEL) for the detection of a possible non-monotonic dose-response curve. Anti-androgenic changes were observable at LOAEL and the clear effect dose level but not at lower exposures. Nipple retention appeared to be the most sensitive measure of anti-androgenic effects, followed by age at sexual maturation, anogenital distance/anogenital index and male sex organ weights, as well as gross and histopathological findings. The results of all five doses indicate the absence of evidence for effects at very low dose levels. A non-monotonic dose-response relationship was not evident for the anti-androgenic drug flutamide.