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.

Appearance of Alveolar Macrophage Subpopulations in Correlation With Histopathological Effects in Short-Term Inhalation Studies With Biopersistent (Nano)Materials.

Following inhalation and deposition in the alveolar region at sufficient dose, biopersistent (nano)materials generally provoke pulmonary inflammation. Alveolar macrophages (AMs) are mediators of pulmonary immune responses and were broadly categorized in pro-inflammatory M1 and anti-inflammatory M2 macrophages. This study aimed at identifying AM phenotype as M1 or M2 upon short-term inhalation exposure to different (nano)materials followed by a postexposure period. Phenotyping of AM was retrospectively performed using immunohistochemistry. M1 (CD68+iNOS+) and M2 (CD68+CD206+ and CD68+ArgI+) AMs were characterized in formalin-fixed, paraffin-embedded lung tissue of rats exposed for 6 hours/day for 5 days to air, 100 mg/m3 nano-TiO2, 25 mg/m3 nano-CeO2, 32 mg/m3 multiwalled carbon nanotubes, or 100 mg/m3 micron-sized quartz. During acute inflammation, relative numbers of M1 AMs were markedly increased, whereas relative numbers of M2 were generally decreased compared to control. Following an exposure-free period, changes in iNOS or CD206 expression correlated with persistence, regression, or progression of inflammation, suggesting a role of M1/M2 AMs in the pathogenesis of pulmonary inflammation. However, no clear correlation of AM subpopulations with qualitatively distinct histopathological findings caused by different (nano)materials was found. A more detailed understanding of the processes underlaying these morphological changes is needed to identify biomarkers for different histopathological outcomes.

Proliferative and Nonproliferative Lesions of the Rat and Mouse Central and Peripheral Nervous Systems: New and Revised INHAND Terms.

Harmonization of diagnostic terminology used during the histopathologic analysis of rodent tissue sections from nonclinical toxicity studies will improve the consistency of data sets produced by laboratories located around the world. The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a cooperative enterprise of 4 major societies of toxicologic pathology to develop a globally accepted standard vocabulary for proliferative and nonproliferative lesions in rodents. A prior manuscript (Toxicol Pathol 2012;40[4 Suppl]:87S-157S) defined multiple diagnostic terms for toxicant-induced lesions, common spontaneous and age-related changes, and principal confounding artifacts in the rat and mouse central nervous system (CNS) and peripheral nervous system (PNS). The current article defines 9 new diagnostic terms and updates 2 previous terms for findings in the rodent CNS and PNS, the need for which has become evident in the years since the publication of the initial INHAND nomenclature for findings in rodent neural tissues. The nomenclature presented in this document is also available electronically on the Internet at the goRENI website (http://www.goreni.org/).

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.

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.

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.

Acetylcholinesterase measurement in various brain regions and muscles of juvenile, adolescent, and adult rats.

Several insecticides, chemicals, and drugs are known to inhibit acetylcholinesterase (AChE) activity, responsible for the cleavage of the neurotransmitter acetylcholine. The administration of AChE inhibitors leads to typical parasympathomimetic (toxic) symptoms in rats. In order to differentiate between compounds acting in various regions of the brain or in peripheral nerves, regulatory authorities demand the measurement of AChE activity in different compartments and the study of potential toxicity at different developmental stages. In the present paper, instructions are given for the necropsy of various regions of the brain depending on rat age. Furthermore, a method validation procedure is described for measuring AChE in these parts of the brain as well as peripheral nerves, serum, and erythrocytes in juvenile, adolescent, and adult rats according to the US EPA method. All investigations were performed within the frame of a regulatory extended one-generation reproductive study (EOGRTS, OECD TG 443). AChE activity increases age dependently in parts of the forebrain (cortex, hippocampus, striatum, but decreases in the mid- and hindbrain (cerebellum, brain stem, medulla oblongata) as well as in peripheral nerves (heart, diaphragm, gastrocnemius muscle). Sex-dependent differences of the AChE activity occur after an age of 11 weeks. The implication of AChE measurement in different brain regions of various age groups is discussed regarding the assessment of AChE inhibitors.

Characterizing "Adversity" of Pathology Findings in Nonclinical Toxicity Studies: Results from the 4th ESTP International Expert Workshop.

The identification of adverse health effects has a central role in the development and risk/safety assessment of chemical entities and pharmaceuticals. There is currently a need for better alignment regarding how nonclinical adversity is determined and characterized. The European Society of Toxicologic Pathology (ESTP) therefore coordinated a workshop to review available definitions of adversity, weigh determining and qualifying factors of adversity based on case examples, and recommend a practical approach to define and characterize adversity in toxicology reports, to serve as a valuable prerequisite for future organ- or lesion-specific workshops planned by the ESTP.

Comparative short-term inhalation toxicity of five organic diketopyrrolopyrrole pigments and two inorganic iron-oxide-based pigments.

Diketopyrrolopyrroles (DPP) are a relatively new class of organic high-performance pigments. The present inhalation and particle characterization studies were performed to compare the effects of five DPP-based pigments (coarse and fine Pigment Red 254, coarse and fine meta-chloro DPP isomer and one form of mixed chlorinated DPP isomers) and compare it to coarse and fine inorganic Pigment Red 101. Wistar rats were exposed head-nose to atmospheres of the respective materials for 6 h/day on 5 consecutive days. Target concentrations were 30 mg/m(3) as high dose for all compounds and selected based occupational exposure limits for respirable nuisance dust. Toxicity was determined after end of exposure and after 3-week recovery using broncho-alveolar lavage fluid (BALF) and microscopic examinations of the entire respiratory tract. Mixed chlorinated DPP isomers and coarse meta-chloro DPP isomer caused marginal changes in BALF, consisting of slight increases of polymorphonuclear neutrophils, and in case of coarse meta-chloro DPP increased MCP-1 and osteopontin levels. Mixed chlorinated DPP isomers, Pigment Red 254, and meta-chloro DPP caused pigment deposits and phagocytosis by alveolar macrophages, slight hypertrophy/hyperplasia of the bronchioles and alveolar ducts, but without evidence of inflammation. In contrast, only pigment deposition and pigment phagocytosis were observed after exposure to Pigment Red 101. All pigments were tolerated well and caused only marginal effects in BALF or no effects at all. Only minor effects were seen on the lung by microscopic examination. There was no evidence of systemic inflammation based on acute-phase protein levels in blood.

Occurrence of Pineal Gland Tumors in Combined Chronic Toxicity/Carcinogenicity Studies in Wistar Rats.

Pineal gland tumors are very rare brain lesions in rats as well as in other species including humans. A total of 8 (out of 1,360 examined) Wistar rats from 3 different combined chronic toxicity/carcinogenicity or mere carcinogenicity studies revealed pineal gland tumors. The tumors were regarded to be spontaneous and unrelated to treatment. The morphology and immunohistochemical evaluation led to the diagnosis malignant pinealoma. The main characteristics that were variably developed within the tumors were the following: cellular atypia, high mitotic index, giant cells, necrosis, Homer Wright rosettes, Flexner-Wintersteiner rosettes and pseudorosettes, positive immunohistochemical reaction for synaptophysin, and neuron-specific enolase. The pineal gland is not a protocol organ for histopathological examination in carcinogenicity studies. Nevertheless, the pineal gland can occasionally be encountered on the routine brain section or if it is the origin of a tumor protruding into the brain, the finding will be recorded. Therefore, although known to be a rare tumor in rats, pineal neoplasms should be included in the list of possible differential diagnoses for brain tumors, especially when the tumor is located in the region of the pineal body.

Applicability of rat precision-cut lung slices in evaluating nanomaterial cytotoxicity, apoptosis, oxidative stress, and inflammation.

The applicability of rat precision-cut lung slices (PCLuS) in detecting nanomaterial (NM) toxicity to the respiratory tract was investigated evaluating sixteen OECD reference NMs (TiO2, ZnO, CeO2, SiO2, Ag, multi-walled carbon nanotubes (MWCNTs)). Upon 24-hour test substance exposure, the PCLuS system was able to detect early events of NM toxicity: total protein, reduction in mitochondrial activity, caspase-3/-7 activation, glutathione depletion/increase, cytokine induction, and histopathological evaluation. Ion shedding NMS (ZnO and Ag) induced severe tissue destruction detected by the loss of total protein. Two anatase TiO2 NMs, CeO2 NMs, and two MWCNT caused significant (determined by trend analysis) cytotoxicity in the WST-1 assay. At non-cytotoxic concentrations, different TiO2 NMs and one MWCNT increased GSH levels, presumably a defense response to reactive oxygen species, and these substances further induced a variety of cytokines. One of the SiO2 NMs increased caspase-3/-7 activities at non-cytotoxic levels, and one rutile TiO2 only induced cytokines. Investigating these effects is, however, not sufficient to predict apical effects found in vivo. Reproducibility of test substance measurements was not fully satisfactory, especially in the GSH and cytokine assays. Effects were frequently observed in negative controls pointing to tissue slice vulnerability even though prepared and handled with utmost care. Comparisons of the effects observed in the PCLuS to in vivo effects reveal some concordances for the metal oxide NMs, but less so for the MWCNT. The highest effective dosages, however, exceeded those reported for rat short-term inhalation studies. To become applicable for NM testing, the PCLuS system requires test protocol optimization.

Biokinetics and effects of barium sulfate nanoparticles.

BACKGROUND: Nanoparticulate barium sulfate has potential novel applications and wide use in the polymer and paint industries. A short-term inhalation study on barium sulfate nanoparticles (BaSO4 NPs) was previously published [Part Fibre Toxicol 11:16, 2014]. We performed comprehensive biokinetic studies of ¹³¹BaSO4 NPs administered via different routes and of acute and subchronic pulmonary responses to instilled or inhaled BaSO4 in rats. METHODS: We compared the tissue distribution of ¹³¹Ba over 28 days after intratracheal (IT) instillation, and over 7 days after gavage and intravenous (IV) injection of ¹³¹BaSO4. Rats were exposed to 50 mg/m³ BaSO4 aerosol for 4 or 13 weeks (6 h/day, 5 consecutive days/week), and then gross and histopathologic, blood and bronchoalveolar lavage (BAL) fluid analyses were performed. BAL fluid from instilled rats was also analyzed. RESULTS: Inhaled BaSO4 NPs showed no toxicity after 4-week exposure, but a slight neutrophil increase in BAL after 13-week exposure was observed. Lung burden of inhaled BaSO4 NPs after 4-week exposure (0.84 ± 0.18 mg/lung) decreased by 95% over 34 days. Instilled BaSO4 NPs caused dose-dependent inflammatory responses in the lungs. Instilled BaSO4 NPs (0.28 mg/lung) was cleared with a half-life of ≈ 9.6 days. Translocated ¹³¹Ba from the lungs was predominantly found in the bone (29%). Only 0.15% of gavaged dose was detected in all organs at 7 days. IV-injected ¹³¹BaSO4 NPs were predominantly localized in the liver, spleen, lungs and bone at 2 hours, but redistributed from the liver to bone over time. Fecal excretion was the dominant elimination pathway for all three routes of exposure. CONCLUSIONS: Pulmonary exposure to instilled BaSO4 NPs caused dose-dependent lung injury and inflammation. Four-week and 13-week inhalation exposures to a high concentration (50 mg/m³) of BaSO4 NPs elicited minimal pulmonary response and no systemic effects. Instilled and inhaled BaSO4 NPs were cleared quickly yet resulted in higher tissue retention than when ingested. Particle dissolution is a likely mechanism. Injected BaSO4 NPs localized in the reticuloendothelial organs and redistributed to the bone over time. BaSO4 NP exhibited lower toxicity and biopersistence in the lungs compared to other poorly soluble NPs such as CeO2 and TiO2.

Application of short-term inhalation studies to assess the inhalation toxicity of nanomaterials.

BACKGROUND: A standard short-term inhalation study (STIS) was applied for hazard assessment of 13 metal oxide nanomaterials and micron-scale zinc oxide. METHODS: Rats were exposed to test material aerosols (ranging from 0.5 to 50 mg/m3) for five consecutive days with 14- or 21-day post-exposure observation. Bronchoalveolar lavage fluid (BALF) and histopathological sections of the entire respiratory tract were examined. Pulmonary deposition and clearance and test material translocation into extra-pulmonary organs were assessed. RESULTS: Inhaled nanomaterials were found in the lung, in alveolar macrophages, and in the draining lymph nodes. Polyacrylate-coated silica was also found in the spleen, and both zinc oxides elicited olfactory epithelium necrosis. None of the other nanomaterials was recorded in extra-pulmonary organs. Eight nanomaterials did not elicit pulmonary effects, and their no observed adverse effect concentrations (NOAECs) were at least 10 mg/m3. Five materials (coated nano-TiO2, both ZnO, both CeO2) evoked concentration-dependent transient pulmonary inflammation. Most effects were at least partially reversible during the post-exposure period.Based on the NOAECs that were derived from quantitative parameters, with BALF polymorphonuclear (PMN) neutrophil counts and total protein concentration being most sensitive, or from the severity of histopathological findings, the materials were ranked by increasing toxic potency into 3 grades: lower toxic potency: BaSO4; SiO2.acrylate (by local NOAEC); SiO2.PEG; SiO2.phosphate; SiO2.amino; nano-ZrO2; ZrO2.TODA; ZrO2.acrylate; medium toxic potency: SiO2.naked; higher toxic potency: coated nano-TiO2; nano-CeO2; Al-doped nano-CeO2; micron-scale ZnO; coated nano-ZnO (and SiO2.acrylate by systemic no observed effect concentration (NOEC)). CONCLUSION: The STIS revealed the type of effects of 13 nanomaterials, and micron-scale ZnO, information on their toxic potency, and the location and reversibility of effects. Assessment of lung burden and material translocation provided preliminary biokinetic information. Based upon the study results, the STIS protocol was re-assessed and preliminary suggestions regarding the grouping of nanomaterials for safety assessment were spelled out.

Time course of lung retention and toxicity of inhaled particles: short-term exposure to nano-Ceria.

Two Ceria nanomaterials (NM-211 and NM-212) were tested for inhalation toxicity and organ burdens in order to design a chronic and carcinogenicity inhalation study (OECD TG No. 453). Rats inhaled aerosol concentrations of 0.5, 5, and 25 mg/m(3) by whole-body exposure for 6 h/day on 5 consecutive days for 1 or 4 weeks with a post-exposure period of 24 or 129 days, respectively. Lungs were examined by bronchoalveolar lavage and histopathology. Inhaled Ceria is deposited in the lung and cleared with a half-time of 40 days; at aerosol concentrations higher than 0.5 mg/m(3), this clearance was impaired resulting in a half-time above 200 days (25 mg/m(3)). After 5 days, Ceria (>0.5 mg/m(3)) induced an early inflammatory reaction by increases of neutrophils in the lung which decreased with time, with sustained exposure, and also after the exposure was terminated (during the post-exposure period). The neutrophil number observed in bronchoalveolar lavage fluid (BALF) was decreasing and supplemented by mononuclear cells, especially macrophages which were visible in histopathology but not in BALF. Further progression to granulomatous inflammation was observed 4 weeks post-exposure. The surface area of the particles provided a dose metrics with the best correlation of the two Ceria's inflammatory responses; hence, the inflammation appears to be directed by the particle surface rather than mass or volume in the lung. Observing the time course of lung burden and inflammation, it appears that the dose rate of particle deposition drove an initial inflammatory reaction by neutrophils. The later phase (after 4 weeks) was dominated by mononuclear cells, especially macrophages. The progression toward the subsequent granulomatous reaction was driven by the duration and amount of the particles in the lung. The further progression of the biological response will be determined in the ongoing long-term study.

Lowest adverse effects concentrations (LOAECs) for formaldehyde exposure.

In 2012 the Committee for Risk Assessment (RAC) of the European Chemicals Agency concluded that 2ppm formaldehyde represent a Lowest Observed Adverse Effect Concentration (LOAEC) for polypoid adenomas, histopathological lesions and cell proliferation. An analysis of all data shows that a LOAEC of 2ppm it is not justified for cell proliferation and polypoid adenomas. Higher values are also supported by a new statistical analysis. For histopathological lesions a NOAEC of 1ppm may be defined but the lesions at 2ppm cannot be regarded as pre-stages for tumour development. One major uncertainty exists: the description of polypoid adenomas and the lesions at 2ppm often is insufficient and diagnostic uncertainties can only be resolved by a re-evaluation according to modern histomorphological standards. Although the discrepancy between our assessment and that of RAC may seem rather small we feel the LOAECs proposed by RAC must be challenged taking into consideration the broad data base for formaldehyde and the potential impact of any published RAC opinion on the present discussions about appropriate occupational and indoor exposure limits.

Mode of action analysis for fluxapyroxad-induced rat liver tumour formation: evidence for activation of the constitutive androstane receptor and assessment of human relevance.

The fungicide fluxapyroxad (BAS 700 F) has been shown to significantly increase the incidence of liver tumours in male Wistar rats at dietary levels of 1500 and 3000 ppm and in female rats at a dietary level of 3000 ppm via a non-genotoxic mechanism. In order to elucidate the mode of action (MOA) for fluxapyroxad-induced rat liver tumour formation a series of in vivo and in vitro investigative studies were undertaken. The treatment of male and female Wistar rats with diets containing 0 (control), 50, 250, 1500 and 3000 ppm fluxapyroxad for 1, 3, 7 and 14 days resulted in a dose-dependent increases in relative weight at 1500 and 3000 ppm from day 3 onwards in both sexes, with an increase in relative liver weight being also observed in male rats given 250 ppm fluxapyroxad for 14 days. Examination of liver sections revealed a centrilobular hepatocyte hypertrophy in some fluxapyroxad treated male and female rats. Hepatocyte replicative DNA synthesis (RDS) was significantly increased in male rats given 1500 and 3000 ppm fluxapyroxad for 3 and 7 days and in female rats given 50-3000 ppm fluxapyroxad for 7 days and 250-3000 ppm fluxapyroxad for 3 and 14 days; the maximal increases in RDS in both sexes being observed after 7 days treatment. The treatment of male and female Wistar rats with 250-3000 ppm fluxapyroxad for 14 days resulted in significant increases in hepatic microsomal total cytochrome P450 (CYP) content and CYP2B subfamily-dependent enzyme activities. Male Wistar rat hepatocytes were treated with control medium and medium containing 1-100 µM fluxapyroxad or 500 µM sodium phenobarbital (NaPB) for 4 days. Treatment with fluxapyroxad and NaPB increased CYP2B and CYP3A enzyme activities and mRNA levels but had little effect on markers of CYP1A and CYP4A subfamily enzymes and of the peroxisomal fatty acid ß-oxidation cycle. Hepatocyte RDS was significantly increased by treatment with fluxapyroxad, NaPB and 25 ng/ml epidermal growth factor (EGF). The treatment of hepatocytes from two male human donors with 1-100 µM fluxapyroxad or 500 µM NaPB for 4 days resulted in some increases in CYP2B and CYP3A enzyme activities and CYP mRNA levels but had no effect on hepatocyte RDS, whereas treatment with EGF resulted in significant increase in RDS in both human hepatocyte preparations. Hepatocytes from male Sprague-Dawley wild type (WT) and constitutive androstane receptor (CAR) knockout (CAR KO) rats were treated with control medium and medium containing 1-16 µM fluxapyroxad or 500 µM NaPB for 4 days. While both fluxapyroxad and NaPB increased CYP2B enzyme activities and mRNA levels in WT hepatocytes, only minor effects were observed in CAR KO rat hepatocytes. Treatment with both fluxapyroxad and NaPB only increased RDS in WT and not in CAR KO rat hepatocytes, whereas treatment with EGF increased RDS in both WT and CAR KO rat hepatocytes. In conclusion, a series of in vivo and in vitro investigative studies have demonstrated that fluxapyroxad is a CAR activator in rat liver, with similar properties to the prototypical CAR activator phenobarbital. A robust MOA for fluxapyroxad-induced rat liver tumour formation has been established. Based on the lack of effect of fluxapyroxad on RDS in human hepatocytes, it is considered that the MOA for fluxapyroxad-induced liver tumour formation is qualitatively not plausible for humans.

Comparative inhalation toxicity of multi-wall carbon nanotubes, graphene, graphite nanoplatelets and low surface carbon black.

BACKGROUND: Carbon nanotubes, graphene, graphite nanoplatelets and carbon black are seemingly chemically identical carbon-based nano-materials with broad technological applications. Carbon nanotubes and carbon black possess different inhalation toxicities, whereas little is known about graphene and graphite nanoplatelets. METHODS: In order to compare the inhalation toxicity of the mentioned carbon-based nanomaterials, male Wistar rats were exposed head-nose to atmospheres of the respective materials for 6 hours per day on 5 consecutive days. Target concentrations were 0.1, 0.5, or 2.5 mg/m3 for multi-wall carbon nanotubes and 0.5, 2.5, or 10 mg/m3 for graphene, graphite nanoplatelets and low-surface carbon black. Toxicity was determined after end of exposure and after three-week recovery using broncho-alveolar lavage fluid and microscopic examinations of the entire respiratory tract. RESULTS: No adverse effects were observed after inhalation exposure to 10 mg/m3 graphite nanoplatelets or relatively low specific surface area carbon black. Increases of lavage markers indicative for inflammatory processes started at exposure concentration of 0.5 mg/m3 for multi-wall carbon nanotubes and 10 mg/m3 for graphene. Consistent with the changes in lavage fluid, microgranulomas were observed at 2.5 mg/m3 multi-wall carbon nanotubes and 10 mg/m3 graphene. In order to evaluate volumetric loading of the lung as the key parameter driving the toxicity, deposited particle volume was calculated, taking into account different methods to determine the agglomerate density. However, the calculated volumetric load did not correlate to the toxicity, nor did the particle surface burden of the lung. CONCLUSIONS: The inhalation toxicity of the investigated carbon-based materials is likely to be a complex interaction of several parameters. Until the properties which govern the toxicity are identified, testing by short-term inhalation is the best option to identify hazardous properties in order to avoid unsafe applications or select safer alternatives for a given application.

Epoxiconazole-induced degeneration in rat placenta and the effects of estradiol supplementation.

Epoxiconazole (CAS-No. 133855-98-8) was recently shown to cause both a marked depletion of maternal estradiol blood levels and a significantly increased incidence of late fetal mortality when administered to pregnant rats throughout gestation (GD 7-18 or 21); estradiol supplementation prevented this epoxiconazole effect in rats (Stinchcombe et al., 2013), indicating that epoxiconazole-mediated estradiol depletion is a critical key event for induction of late fetal resorptions in rats. For further elucidation of the mode of action, the placentas from these modified prenatal developmental toxicity experiments with 23 and 50 mg/kg bw/d epoxiconazole were subjected to a detailed histopathological examination. This revealed dose-dependent placental degeneration characterized by cystic dilation of maternal sinuses in the labyrinth, leading to rupture of the interhemal membrane. Concomitant degeneration occurred in the trophospongium. Both placentas supporting live fetuses and late fetal resorptions were affected; the highest degree of severity was observed in placentas with late resorptions. Placental degeneration correlated with a severe decline in maternal serum estradiol concentration. Supplementation with 0.5 and 1.0 µg of the synthetic estrogen estradiol cyclopentylpropionate per day reduced the severity of the degeneration in placentas with live fetuses. The present study demonstrates that both the placental degeneration and the increased incidence of late fetal resorptions are due to decreased levels of estrogen, since estrogen supplementation ameliorates the former and abolishes the latter.

Species differences in developmental toxicity of epoxiconazole and its relevance to humans.

Epoxiconazole, a triazole-based fungicide, was tested in toxicokinetic, prenatal and pre-postnatal toxicity studies in guinea pigs, following oral (gavage) administration at several dose levels (high dose: 90 mg/kg body weight per day). Maternal toxicity was evidenced by slightly increased abortion rates and by histopathological changes in adrenal glands, suggesting maternal stress. No compound-related increase in the incidence of malformations or variations was observed in the prenatal study. In the pre-postnatal study, epoxiconazole did not adversely affect gestation length, parturition, or postnatal growth and development. Administration of epoxiconazole did not alter circulating estradiol levels. Histopathological examination of the placentas did not reveal compound-related effects. The results in guinea pigs are strikingly different to those observed in pregnant rats, in which maternal estrogen depletion, pathological alteration of placentas, increased gestation length, late fetal death, and dystocia were observed after administration of epoxiconazole. In the studies reported here, analysis of maternal plasma concentrations and metabolism after administration of radiolabeled epoxiconazole demonstrated that the different results in rats and guinea pigs were not due to different exposures of the animals. A comprehensive comparison of hormonal regulation of pregnancy and birth in murid rodents and primates indicates that the effects on pregnancy and parturition observed in rats are not applicable to humans. In contrast, the pregnant guinea pig shares many similarities to pregnant humans regarding hormonal regulation and is therefore considered to be a suitable species for extrapolation of related effects to humans.

Effects of estrogen coadministration on epoxiconazole toxicity in rats.

Epoxiconazole (EPX; CAS-No. 133855-98-8) is a triazole class-active substance of plant protection products. At a dose level of 50 mg/kg bw/day, it causes a significantly increased incidence of late fetal mortality when administered to pregnant rats throughout gestation (gestation day [GD] 7-18 or 21), as reported previously (Taxvig et al., 2007, 2008) and confirmed in these studies. Late fetal resorptions occurred in the presence of significant maternal toxicity such as clear reduction of corrected body weight gain, signs of anemia, and, critically, a marked reduction of maternal estradiol plasma levels. Furthermore, estradiol supplementation at dose levels of 0.5 or 1.0 µg/animal/day of estradiol cyclopentylpropionate abolished the EPX-mediated late fetal resorptions. No increased incidences of external malformations were found in rats cotreated with 50 mg/kg bw/day EPX and estradiol cyclopentylpropionate, indicating that the occurrence of malformations was not masked by fetal mortality under the study conditions. Overall, the study data indicate that fetal mortality observed in rat studies with EPX is not the result of direct fetal toxicity but occurs indirectly via depletion of maternal estradiol levels. The clarification of the human relevance of the estrogen-related mechanism behind EPX-mediated late fetal resorptions in rats warrants further studies. In particular, this should involve investigation of the placenta (Rey Moreno et al., 2013), since it is the materno-fetal interface and crucial for fetal maintenance. The human relevance is best addressed in a species which is closer to humans with reference to placentation and hormonal regulation of pregnancy, such as the guinea pig (Schneider et al., 2013).