Connecting Gut Microbial Diversity with Plasma Metabolome and Fecal Bile Acid Changes Induced by the Antibiotics Tobramycin and Colistin Sulfate.

The diversity of microbial species in the gut has a strong influence on health and development of the host. Further, there are indications that the variation in expression of gut bacterial metabolic enzymes is less diverse than the taxonomic profile, underlying the importance of microbiome functionality, particularly from a toxicological perspective. To address these relationships, the gut bacterial composition of Wistar rats was altered by a 28 day oral treatment with the antibiotics tobramycin or colistin sulfate. On the basis of 16S marker gene sequencing data, tobramycin was found to cause a strong reduction in the diversity and relative abundance of the microbiome, whereas colistin sulfate had only a marginal impact. Associated plasma and fecal metabolomes were characterized by targeted mass spectrometry-based profiling. The fecal metabolome of tobramycin-treated animals had a high number of significant alterations in metabolite levels compared to controls, particularly in amino acids, lipids, bile acids (BAs), carbohydrates, and energy metabolites. The accumulation of primary BAs and significant reduction of secondary BAs in the feces indicated that the microbial alterations induced by tobramycin inhibit bacterial deconjugation reactions. The plasma metabolome showed less, but still many alterations in the same metabolite groups, including reductions in indole derivatives and hippuric acid, and furthermore, despite marginal effects of colistin sulfate treatment, there were nonetheless systemic alterations also in BAs. Aside from these treatment-based differences, we also uncovered interindividual differences particularly centering on the loss of Verrucomicrobiaceae in the microbiome, but with no apparent associated metabolite alterations. Finally, by comparing the data set from this study with metabolome alterations in the MetaMapTox database, key metabolite alterations were identified as plasma biomarkers indicative of altered gut microbiomes resulting from a wide activity spectrum of antibiotics.

Towards a science-based testing strategy to identify maternal thyroid hormone imbalance and neurodevelopmental effects in the progeny - Part IV: the ECETOC and CLE Proposal for a Thyroid Function-Related Neurodevelopmental Toxicity Testing and Assessment Scheme (Thyroid-NDT-TAS).

Following the European Commission Endocrine Disruptor Criteria, substances shall be considered as having endocrine disrupting properties if they (a) elicit adverse effects, (b) have endocrine activity, and (c) the two are linked by an endocrine mode-of-action (MoA) unless the MoA is not relevant for humans. A comprehensive, structured approach to assess whether substances meet the Endocrine Disruptor Criteria for the thyroid modality (EDC-T) is currently unavailable. Here, the European Centre for Ecotoxicology and Toxicology of Chemicals Thyroxine Task Force and CropLife Europe propose a Thyroid Function-Related Neurodevelopmental Toxicity Testing and Assessment Scheme (Thyroid-NDT-TAS). In Tier 0, before entering the Thyroid-NDT-TAS, all available in vivo, in vitro and in silico data are submitted to weight-of-evidence (WoE) evaluations to determine whether the substance of interest poses a concern for thyroid disruption. If so, Tier 1 of the Thyroid-NDT-TAS includes an initial MoA and human relevance assessment (structured by the key events of possibly relevant adverse outcome pathways) and the generation of supportive in vitro/in silico data, if relevant. Only if Tier 1 is inconclusive, Tier 2 involves higher-tier testing to generate further thyroid- and/or neurodevelopment-related data. Tier 3 includes the final MoA and human relevance assessment and an overarching WoE evaluation to draw a conclusion on whether, or not, the substance meets the EDC-T. The Thyroid-NDT-TAS is based on the state-of-the-science, and it has been developed to minimise animal testing. To make human safety assessments more accurate, it is recommended to apply the Thyroid-NDT-TAS during future regulatory assessments.

A high-throughput metabolomics in vitro platform for the characterization of hepatotoxicity.

Cell-based metabolomics provides multiparametric physiologically relevant readouts that can be highly advantageous for improved, biologically based decision making in early stages of compound development. Here, we present the development of a 96-well plate LC-MS/MS-based targeted metabolomics screening platform for the classification of liver toxicity modes of action (MoAs) in HepG2 cells. Different parameters of the workflow (cell seeding density, passage number, cytotoxicity testing, sample preparation, metabolite extraction, analytical method, and data processing) were optimized and standardized to increase the efficiency of the testing platform. The applicability of the system was tested with seven substances known to be representative of three different liver toxicity MoAs (peroxisome proliferation, liver enzyme induction, and liver enzyme inhibition). Five concentrations per substance, aimed at covering the complete dose-response curve, were analyzed and 221 uniquely identified metabolites were measured, annotated, and allocated in 12 different metabolite classes such as amino acids, carbohydrates, energy metabolism, nucleobases, vitamins and cofactors, and diverse lipid classes. Multivariate and univariate analyses showed a dose response of the metabolic effects, a clear differentiation between liver toxicity MoAs and resulted in the identification of metabolite patterns specific for each MoA. Key metabolites indicative of both general and mechanistic specific hepatotoxicity were identified. The method presented here offers a multiparametric, mechanistic-based, and cost-effective hepatotoxicity screening that provides MoA classification and sheds light into the pathways involved in the toxicological mechanism. This assay can be implemented as a reliable compound screening platform for improved safety assessment in early compound development pipelines.

Application of high throughput in vitro metabolomics for hepatotoxicity mode of action characterization and mechanistic-anchored point of departure derivation: a case study with nitrofurantoin.

Omics techniques have been increasingly recognized as promising tools for Next Generation Risk Assessment. Targeted metabolomics offer the advantage of providing readily interpretable mechanistic information about perturbed biological pathways. In this study, a high-throughput LC-MS/MS-based broad targeted metabolomics system was applied to study nitrofurantoin metabolic dynamics over time and concentration and to provide a mechanistic-anchored approach for point of departure (PoD) derivation. Upon nitrofurantoin exposure at five concentrations (7.5 µM, 15 µM, 20 µM, 30 µM and 120 µM) and four time points (3, 6, 24 and 48 h), the intracellular metabolome of HepG2 cells was evaluated. In total, 256 uniquely identified metabolites were measured, annotated, and allocated in 13 different metabolite classes. Principal component analysis (PCA) and univariate statistical analysis showed clear metabolome-based time and concentration effects. Mechanistic information evidenced the differential activation of cellular pathways indicative of early adaptive and hepatotoxic response. At low concentrations, effects were seen mainly in the energy and lipid metabolism, in the mid concentration range, the activation of the antioxidant cellular response was evidenced by increased levels of glutathione (GSH) and metabolites from the de novo GSH synthesis pathway. At the highest concentrations, the depletion of GSH, together with alternations reflective of mitochondrial impairments, were indicative of a hepatotoxic response. Finally, a metabolomics-based PoD was derived by multivariate PCA using the whole set of measured metabolites. This approach allows using the entire dataset and derive PoD that can be mechanistically anchored to established key events. Our results show the suitability of high throughput targeted metabolomics to investigate mechanisms of hepatoxicity and derive point of departures that can be linked to existing adverse outcome pathways and contribute to the development of new ones.

Analysis of health concerns not addressed by REACH for low tonnage chemicals and opportunities for new approach methodology.

In Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) the criterion for deciding the studies that must be performed is the annual tonnage of the chemical manufactured or imported into the EU. The annual tonnage may be considered as a surrogate for levels of human exposure but this does not take into account the physico-chemical properties and use patterns that determine exposure. Chemicals are classified using data from REACH under areas of health concern covering effects on the skin and eye; sensitisation; acute, repeated and prolonged systemic exposure; effects on genetic material; carcinogenicity; and reproduction and development. We analysed the mandated study lists under REACH for each annual tonnage band in terms of the information they provide on each of the areas of health concern. Using the European Chemicals Agency (ECHA) REACH Registration data base of over 20,000 registered substances, we found that only 19% of registered substances have datasets on all areas of health concern. Information limited to acute exposure, sensitisation and genotoxicity was found for 62%. The analysis highlighted the shortfall of information mandated for substances in the lower tonnage bands. Deploying New Approach Methodologies (NAMs) at this lower tonnage band to assess health concerns which are currently not covered by REACH, such as repeat and extended exposure and carcinogenicity, would provide additional information and would be a way for registrants and regulators to gain experience in the use of NAMs. There are currently projects in Europe aiming to develop NAM-based assessment frameworks and they could find their first use in assessing low tonnage chemicals once confidence has been gained by their evaluation with data rich chemicals.

Towards a science-based testing strategy to identify maternal thyroid hormone imbalance and neurodevelopmental effects in the progeny-part III: how is substance-mediated thyroid hormone imbalance in pregnant/lactating rats or their progeny related to neurodevelopmental effects?

This review investigated which patterns of thyroid- and brain-related effects are seen in rats upon gestational/lactational exposure to 14 substances causing thyroid hormone imbalance by four different modes-of-action (inhibition of thyroid peroxidase, sodium-iodide symporter and deiodinase activities, enhancement of thyroid hormone clearance) or to dietary iodine deficiency. Brain-related parameters included motor activity, cognitive function, acoustic startle response, hearing function, periventricular heterotopia, electrophysiology and brain gene expression. Specific modes-of-action were not related to specific patterns of brain-related effects. Based upon the rat data reviewed, maternal serum thyroid hormone levels do not show a causal relationship with statistically significant neurodevelopmental effects. Offspring serum thyroxine together with offspring serum triiodothyronine and thyroid stimulating hormone appear relevant to predict the likelihood for neurodevelopmental effects. Based upon the collated database, thresholds of ≥60%/≥50% offspring serum thyroxine reduction and ≥20% and statistically significant offspring serum triiodothyronine reduction indicate an increased likelihood for statistically significant neurodevelopmental effects; accuracies: 83% and 67% when excluding electrophysiology (and gene expression). Measurements of brain thyroid hormone levels are likely relevant, too. The extent of substance-mediated thyroid hormone imbalance appears more important than substance mode-of-action to predict neurodevelopmental impairment in rats. Pertinent research needs were identified, e.g. to determine whether the phenomenological offspring thyroid hormone thresholds are relevant for regulatory toxicity testing. The insight from this review shall be used to suggest a tiered testing strategy to determine whether gestational/lactational substance exposure may elicit thyroid hormone imbalance and potentially also neurodevelopmental effects.

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.

In vitro models to detect in vivo bile acid changes induced by antibiotics.

Bile acid homeostasis plays an important role in many biological activities through the bile-liver-gut axis. In this study, two in vitro models were applied to further elucidate the mode of action underlying reported in vivo bile acid changes induced by antibiotics (colistin sulfate, tobramycin, meropenem trihydrate, and doripenem hydrate). 16S rRNA analysis of rat fecal samples anaerobically incubated with these antibiotics showed that especially tobramycin induced changes in the gut microbiota. Furthermore, tobramycin was shown to inhibit the microbial deconjugation of taurocholic acid (TCA) and the transport of TCA over an in vitro Caco-2 cell layer used as a model to mimic intestinal bile acid reuptake. The effects induced by the antibiotics in the in vitro model systems provide novel and complementary insight explaining the effects of the antibiotics on microbiota and fecal bile acid levels upon 28-day in vivo treatment of rats. In particular, our results provide insight in the mode(s) of action underlying the increased levels of TCA in the feces upon tobramycin exposure. Altogether, the results of the present study provide a proof-of-principle on how in vitro models can be used to elucidate in vivo effects on bile acid homeostasis, and to obtain insight in the mode(s) of action underlying the effect of an antibiotic, in this case tobramycin, on bile acid homeostasis via effects on intestinal bile acid metabolism and reuptake.

A framework for chemical safety assessment incorporating new approach methodologies within REACH.

The long-term investment in new approach methodologies (NAMs) within the EU and other parts of the world is beginning to result in an emerging consensus of how to use information from in silico, in vitro and targeted in vivo sources to assess the safety of chemicals. However, this methodology is being adopted very slowly for regulatory purposes. Here, we have developed a framework incorporating in silico, in vitro and in vivo methods designed to meet the requirements of REACH in which both hazard and exposure can be assessed using a tiered approach. The outputs from each tier are classification categories, safe doses, and risk assessments, and progress through the tiers depends on the output from previous tiers. We have exemplified the use of the framework with three examples. The outputs were the same or more conservative than parallel assessments based on conventional studies. The framework allows a transparent and phased introduction of NAMs in chemical safety assessment and enables science-based safety decisions which provide the same level of public health protection using fewer animals, taking less time, and using less financial and expert resource. Furthermore, it would also allow new methods to be incorporated as they develop through continuous selective evolution rather than periodic revolution.

Towards a science-based testing strategy to identify maternal thyroid hormone imbalance and neurodevelopmental effects in the progeny - part II: how can key events of relevant adverse outcome pathways be addressed in toxicological assessments?

The current understanding of thyroid-related adverse outcome pathways (AOPs) with adverse neurodevelopmental outcomes in mammals has been reviewed. This served to establish if standard rodent toxicity test methods and in vitro assays allow identifying thyroid-related modes-of-action potentially leading to adverse neurodevelopmental outcomes, and the human relevance of effects - in line with the European Commission's Endocrine Disruptor Criteria. The underlying hypothesis is that an understanding of the key events of relevant AOPs provides insight into differences in incidence, magnitude, or species sensitivity of adverse outcomes. The rodent studies include measurements of serum thyroid hormones, thyroid gland pathology and neurodevelopmental assessments, but do not directly inform on specific modes-of-action. Opportunities to address additional non-routine parameters reflecting critical events of AOPs in toxicological assessments are presented. These parameters appear relevant to support the identification of specific thyroid-related modes-of-action, provided that prevailing technical limitations are overcome. Current understanding of quantitative key event relationships is often weak, but would be needed to determine if the triggering of a molecular initiating event will ultimately result in an adverse outcome. Also, significant species differences in all processes related to thyroid hormone signalling are evident, but the biological implications thereof (including human relevance) are often unknown. In conclusion, careful consideration of the measurement (e.g. timing, method) and interpretation of additional non-routine parameters is warranted. These findings will be used in a subsequent paper to propose a testing strategy to identify if a substance may elicit maternal thyroid hormone imbalance and potentially also neurodevelopmental effects in the progeny.

A Workflow for the Performance of the Differential Ovarian Follicle Count Using Deep Neuronal Networks.

In order to automate the counting of ovarian follicles required in multigeneration reproductive studies performed in the rat according to Organization for Economic Co-operation and Development guidelines 443 and 416, the application of deep neural networks was tested. The manual evaluation of the differential ovarian follicle count is a tedious and time-consuming task that requires highly trained personnel. In this regard, deep learning outputs provide overlay pictures for a more detailed documentation, together with an increased reproducibility of the counts. To facilitate the planned good laboratory practice (GLP) validation a workflow was set up using MLFlow to make all steps from generating of scans, training of the neural network, uploading of study images to the neural network, generation and storage of the results in a compliant manner controllable and reproducible. PyTorch was used as main framework to build the Faster region-based convolutional neural network for the training. We compared the performances of different depths of ResNet models with specific regard to the sensitivity, specificity, accuracy of the models. In this paper, we describe all steps from data labeling, training of networks, and the performance metrics chosen to evaluate different network architectures. We also make recommendation on steps, which should be taken into consideration when GLP validation is aimed for.

Novel testing strategy for prediction of rat biliary excretion of intravenously administered estradiol-17ß glucuronide.

The aim of the present study was to develop a generic rat physiologically based kinetic (PBK) model that includes a novel testing strategy where active biliary excretion is incorporated using estradiol-17ß glucuronide (E217ßG) as the model substance. A major challenge was the definition of the scaling factor for the in vitro to in vivo conversion of the PBK-model parameter Vmax. In vitro values for the Vmax and Km for transport of E217ßG were found in the literature in four different studies based on experiments with primary rat hepatocytes. The required scaling factor was defined based on fitting the PBK model-based predicted values to reported experimental data on E217ßG blood levels and cumulative biliary E217ßG excretion. This resulted in a scaling factor of 129 mg protein/g liver. With this scaling factor the PBK model predicted the in vivo data for blood and cumulative biliary E217ßG levels with on average of less than 1.8-fold deviation. The study provides a proof of principle on how biliary excretion can be included in a generic PBK model using primary hepatocytes to define the kinetic parameters that describe the biliary excretion.

Toward a science-based testing strategy to identify maternal thyroid hormone imbalance and neurodevelopmental effects in the progeny - part I: which parameters from human studies are most relevant for toxicological assessments?

The 2018 European Food Safety Authority/European Chemicals Agency Guidance on the Identification of Endocrine Disruptors lacks clarity on how the presence or absence of substance-induced maternal thyroid hormone imbalance, or the potential for subsequent deleterious consequences in child neurodevelopment, should be established by toxicological assessments. To address these uncertainties, this narrative review evaluates human evidence on how altered maternal thyroid function may be associated with child neurodevelopmental outcomes; and seeks to identify parameters in human studies that appear most relevant for toxicological assessments. Serum levels of free thyroxine (fT4) and thyroid stimulating hormone (TSH) are most frequently measured when assessing thyroid function in pregnant women, whereas a broad spectrum of neurodevelopmental parameters is used to evaluate child neurodevelopment. The human data confirms an association between altered maternal serum fT4 and/or TSH and increased risk for child neurodevelopmental impairment. Quantitative boundaries of effects indicative of increased risks need to be established. Moreover, it is unknown if altered serum levels of total T4, free or total triiodothyronine, or parameters unrelated to serum thyroid hormones might be more relevant indicators of such effects. None of the human studies established a link between substance-mediated liver enzyme induction and increased serum thyroid hormone clearance, let alone further to child neurodevelopmental impairment. This review identifies research needs to contribute to the development of toxicity testing strategies, to reliably predict whether substances have the potential to impair child neurodevelopment via maternal thyroid hormone imbalance.

Xenobiotica-metabolizing enzyme induction potential of chemicals in animal studies: NanoString nCounter gene expression and peptide group-specific immunoaffinity as accelerated and economical substitutions for enzyme activity determinations?

Xenobiotica-metabolizing enzyme (XME) induction is a relevant biological/biochemical process vital to understanding the toxicological profile of xenobiotics. Early recognition of XME induction potential of compounds under development is therefore important, yet its determination by traditional XME activity measurements is time consuming and cost intensive. A proof-of-principle study was therefore designed due to the advent of faster and less cost-intensive methods for determination of enzyme protein and transcript levels to determine whether two such methods may substitute for traditional measurement of XME activity determinations. The results of the study show that determination of enzyme protein levels by peptide group-specific immunoaffinity enrichment/MS and/or determination of gene expression by NanoString nCounter may serve as substitutes for traditional evaluation methodology and/or as an early predictor of potential changes in liver enzymes. In this study, changes of XME activity by the known standard XME inducers phenobarbital, beta-naphthoflavone and Aroclor 1254 were demonstrated by these two methods. To investigate the applicability of these methods to demonstrate XME-inducing activity of an unknown, TS was also examined and found to be an XME inducer. More specifically, TS was found to be a phenobarbital-type inducer (likely mediated by CAR rather than PXR as nuclear receptor), but not due to Ah receptor-mediated or antioxidant response element-mediated beta-naphthoflavone-type induction. The results for TS were confirmed via enzymatic activity measurements. The results of the present study demonstrate the potential applicability of NanoString nCounter mRNA quantitation and peptide group-specific immunoaffinity enrichment/MS protein quantitation for predicting compounds under development to be inducers of liver XME activity.

In vitro-to-in vivo extrapolation (IVIVE) by PBTK modeling for animal-free risk assessment approaches of potential endocrine-disrupting compounds.

While in vitro testing is used to identify hazards of chemicals, nominal in vitro assay concentrations may misrepresent potential in vivo effects and do not provide dose-response data which can be used for a risk assessment. We used reverse dosimetry to compare in vitro effect concentrations-to-in vivo doses causing toxic effects related to endocrine disruption. Ten compounds (acetaminophen, bisphenol A, caffeine, 17α-ethinylestradiol, fenarimol, flutamide, genistein, ketoconazole, methyltestosterone, and trenbolone) have been tested in the yeast estrogen screening (YES) or yeast androgen-screening (YAS) assays for estrogen and androgen receptor binding, as well as the H295R assay (OECD test guideline no. 456) for potential interaction with steroidogenesis. With the assumption of comparable concentration-response ratios of these effects in the applied in vitro systems and the in vivo environment, the lowest observed effect concentrations from these assays were extrapolated to oral doses (LOELs) by reverse dosimetry. For extrapolation, an eight-compartment Physiologically Based Toxicokinetic (PBTK) rat model based on in vitro and in silico input data was used. The predicted LOEL was then compared to the LOEL actually observed in corresponding in vivo studies (YES/YAS assay versus uterotrophic or Hershberger assay and steroidogenesis assay versus pubertal assay or generation studies). This evaluation resulted in 6 out of 10 compounds for which the predicted LOELs were in the same order of magnitude as the actual in vivo LOELs. For four compounds, the predicted LOELs differed by more than tenfold from the actual in vivo LOELs. In conclusion, these data demonstrate the applicability of reverse dosimetry using a simple PBTK model to serve in vitro-in silico-based risk assessment, but also identified cases and test substance were the applied methods are insufficient.

The in vivo developmental toxicity of diethylstilbestrol (DES) in rat evaluated by an alternative testing strategy.

In the present study, we evaluated an alternative testing strategy to quantitatively predict the in vivo developmental toxicity of the synthetic hormone diethylstilbestrol (DES). To this end, a physiologically based kinetic (PBK) model was defined that was subsequently used to translate concentration-response data for the in vitro developmental toxicity of DES, obtained in the ES-D3 cell differentiation assay, into predicted in vivo dose-response data for developmental toxicity. The previous studies showed that the PBK model-facilitated reverse dosimetry approach is a useful approach to quantitatively predict the developmental toxicity of several developmental toxins. The results obtained in the present study show that the PBK model adequately predicted DES blood concentrations in rats. Further studies revealed that DES tested positive in the ES-D3 differentiation assay and that DES-induced inhibition of the ES-D3 cell differentiation could be counteracted by the estrogen receptor alpha (ERα) antagonist fulvestrant, indicating that the in vitro ES-D3 cell differentiation assay was able to mimic the role of ERα reported in the mode of action underlying the developmental toxicity of DES in vivo. In spite of this, combining these in vitro data with the PBK model did not adequately predict the in vivo developmental toxicity of DES in a quantitative way. It is concluded that although the EST qualifies DES as a developmental toxin and detects the role of ERα in this process, the ES-D3 cell differentiation assay of the EST apparently does not adequately capture the processes underlying DES-induced developmental toxicity in vivo.

Variance of body and organ weights in 28-day studies in mice.

The OECD guideline 407 outlines the conduct of 28-day studies in rodents to detect systemic toxicity with focus on endocrine and immunotoxic effects. It was validated with the rat as preferred model species. Justification is required for other rodent species, as an increased variability is expected compared to the rat. We investigated the variability of organ weights in the mouse and compared this to data published for the rat in the validation report of test guideline 407. Furthermore, the influence of the immunotoxic model substance cyclophosphamide on spleen and thymus weights in the mouse in immunotoxicity studies (duration 28 days) is reported and discussed, an immunotoxic model substance was not included in the validation report. Historical control data were compiled for mouse studies performed according to OECD 407 and for immunotoxicity studies between 2008 and 2013 at BASF SE. For absolute weights, the coefficient of variation was determined for each study group and compared with the rat. Adrenal glands, ovaries and to lesser degree testes and prostate showed higher coefficients of variation in the mouse (most pronounced in adrenal glands in male animals: rat 5%-17%, CD1 mouse 20%-51%). Effects of cyclophosphamide were best detected measuring the thymus weight.

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.

Towards a generic physiologically based kinetic model to predict in vivo uterotrophic responses in rats by reverse dosimetry of in vitro estrogenicity data.

Physiologically based kinetic (PBK) modelling-based reverse dosimetry is a promising tool for the prediction of in vivo developmental toxicity using in vitro concentration-response data. In the present study, the potential of this approach to predict the dose-dependent increase of uterus weight in rats upon exposure to estrogenic chemicals was assessed. In vitro concentration-response data of 17ß-estradiol (E2) and bisphenol A (BPA) obtained in the MCF-7/BOS proliferation assay, the U2OS ER-CALUX assay and the yeast estrogen screen (YES) assay, were translated into in vivo dose-response data in rat, using a PBK model with a minimum number of in vitro and in silico determined parameter values. To evaluate the predictions made, benchmark dose (BMD) analysis was performed on the predicted dose-response data and the obtained BMDL10 values were compared with BMDL10 values derived from data on the effects of E2 and BPA in the uterotrophic assay reported in the literature. The results show that predicted dose-response data of E2 and BPA matched with the data from in vivo studies when predictions were made based on YES assay data. The YES assay-based predictions of the BMDL10 values differed 3.9-fold (E2) and 4.7- to 13.4-fold (BPA) from the BMDL10 values obtained from the in vivo data. The present study provides the proof-of-principle that PBK modelling-based reverse dosimetry of YES assay data using a minimum PBK model can predict dose-dependent in vivo uterus growth caused by estrogenic chemicals. In future studies, the approach should be extended to include other estrogens.

Uterine adenocarcinoma in the rat induced by afidopyropen. An analysis of the lesion's induction, progression and its relevance to humans.

Afidopyropen is a novel insecticide that acts as a TRPV channel modulator in chordotonal organs of target insects. In two carcinogenicity studies with Fischer rats, an increased incidence of uterine adenocarcinomas was observed at 1000 and 3000 ppm. This finding prompted an investigation of the mechanism of the tumor formation as well as the relevance of this mechanism to humans. The mechanistic work took parallel paths: one path investigated the pharmacokinetic properties of the test substance at the doses where the tumors were found; while the second path examined the key mechanistic events that culminated in uterine adenocarcinomas. The results of the investigation indicated that the tumors only occurred at doses where excretion of test substance was saturated - indicating that homeostatic biological and/or physiological processes were overwhelmed. At the doses where these processes were overwhelmed, the test substance acted through a mechanism of dopamine agonism, triggering a cascade key events that resulted in uterine adenocarcinomas. An analysis of these mechanisms observed in rat showed that they are both quantitatively (pharmacokinetic mechanism) and qualitatively (dopamine agonism mechanism) not relevant to humans. Therefore the uterine adenocarcinomas observed in the rat associated with high doses of Afidopyropen are not expected to pose a carcinogenic risk to humans.