Placental disease and abnormal umbilical artery Doppler waveforms in Trisomy 21 Pregnancy: A case-control study

Abstracts / Reproductive Toxicology 64 (2016) 29–49

P-41 Teratogenicity of a widely used glyphosate-based herbicide formulation in Xenopus laevis Melissa Saibene 1,∗ , Anita Colombo 1 , Patrizia Bonfanti 1 , Rosalba Gornati 2 , Giovanni Bernardini 2 , Renato Bacchetta 3 , Paride Mantecca 1 1

Department Earth and Environmental Sciences, Research Centre POLARIS, University of Milano Bicocca, Milan, Italy 2 Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy 3 Department of Biosciences, University of Milan, Milan, Italy Glyphosate is an Active Ingredient (AI) largely employed in broad-spectrum herbicide commercial formulations, widely used in agriculture, the domestic area and in aquatic weed control. Glyphosate-based herbicides are a growing market largely due to the corresponding increase of cultivations of Glyphosate-tolerant transgenic crops in several countries. However, the toxicity of Gbased herbicides (G) against non-target organisms is still debated even though different effects including oxidative stress, mutagenesis, reproductive toxicity and teratogenicity have been reported. In this study we tested the effects of Roundup Power 2.0® , a next-generation formulation of G on the embryonic development of Xenopus laevis using the Frog Embryo Teratogenesis Assay-Xenopus (FETAX). Amphibians are a good indicator for aquatic pollution and they can represent an indirect target of agrochemical exposure as shown by the increasing mortality rates of these organism during last decade. Our results showed that Roundup Power 2.0® caused lethality only at high concentrations, while sub-lethal doses (96-h TC50 7.3 mg/L) were enough to induce concentration-dependent malformations in larvae, such as microphthalmia, oval shaped eyes, craniofacial defects and abnormal gut coiling. The commercial formulation is a complex mixture of AI and adjuvants, that improve penetration through plant surface to the site of action. For this reason, comparison of the effects of the commercial formulations with the effects caused by pure Glyphosate is warranted. Further experiments to better characterize Roundup Power 2.0® - and Glyphosate-induced malformations, using histological, ultrastructural and gene expression analysis will be performed. It is expected that the results from this work will increase our knowledge regarding the developmental effects of Glyphosate-based herbicides and thus aid in the production of safer formulations that are equally effective in weed control. http://dx.doi.org/10.1016/j.reprotox.2016.06.102 P-42 Standardisation and design of study on developmental thyroid toxicity Deepak G. Ujawane, Kishor C. Hadiya ∗ , Mukesh P. Poshiya, Jay P. Rabadia, Foram K. Parikh, Jaydip T. Mistry, Manish P. Patel Jai Research Foundation, Vapi, Gujarat, India Introduction: Thyroid homeostasis plays a crucial role in growth, metabolism, and neurocognitive development. Imbalances during in utero development may have irreversible consequences on the growth and the brain development. Numerous pesticide

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chemicals perturb thyroid hormone homeostasis via reduction of circulating thyroid hormones. Methods: There are no firm guidelines to draw conclusion, whether a chemical is a thyroid toxicant or not. The aim of the present study was to establish specific time-points/endpoints which can be used as a reference for conducting specific thyroid developmental toxicity studies or included in various reproductive/development studies. In this study, parent female animals were administered, by oral gavage, 6-propyl-2-thiouracil during gestation and lactation period. Pups were treated through oral gavage from PND-8 to 21. Pooled blood (per litter) was collected from GD-20 fetuses, and pups/sex of PND-4. Blood was collected from dams on GD-20 and LD-22. T4, TSH, and T3 hormonal analysis was carried out as per the procedure given in ELISA kit. Liver, kidney, brain, spleen, and thyroid weight was taken from GD-20 & LD22 dams and LD-22 pups/sex. Microscopic examination of thyroid (with parathyroid) was conducted in fetus, pups/sex (of PND-4 & 22), and dams (on GD-20 & LD-22). Results: Significant decreases were seen in serum thyroid hormones (decrease in T4, T3, and increase in TSH) in parent animals sacrificed on GD-20, LD-22 and in fetuses (of GD-20), in pups of PND-4 and PND-22. A significant decrease in body weight and increase in thyroid (with parathyroid) weight was observed in parents, fetuses, and pups. Follicular cell hypertrophy and depletion of colloid in the follicular lumen was observed in fetuses. Whereas in parent animals and in pups of PND-4 & 22, follicular cell hypertrophy, depletion of colloid in the follicular lumen, decreased follicular lumen size, and hyperplasia of follicular cells were observed. Conclusions: The various time-points for determination of the thyroid hormone level and evaluation of the microscopy on thyroid (with parathyroid) play a vital role for identifying the chemicals with positive effects. http://dx.doi.org/10.1016/j.reprotox.2016.06.103 P-43 Placental disease and abnormal umbilical artery Doppler waveforms in Trisomy 21 Pregnancy: A case-control study Fionnuala Mone 1,2,∗ , Edward Corry 3 , Ricardo Segurado 4 , Paul Downey 5 , Peter McParland 2 , Fionnuala M. McAuliffe 1,2 , Eoghan E. Mooney 5 1

UCD Obstetrics and Gynaecology, School of Medicine and Medical Science, University College Dublin, National Maternity Hospital, Dublin, Ireland 2 Department of Fetal Medicine, National Maternity Hospital, Dublin, Ireland 3 Department of Obstetrics and Gynaecology, National Maternity Hospital, Dublin, Ireland 4 CSTAR, School of Public Health, Physiotherapy and Population Science, University College Dublin, Ireland 5 Department of Pathology and Laboratory Medicine, National Maternity Hospital, Dublin, Ireland Introduction: The objectives of this study were firstly to determine the proportion of placental pathology in fetuses affected by trisomy 21 (T21) using current pathological descriptive terminology and secondly to examine if a correlation existed between the finding of an abnormal umbilical artery Doppler (UAD) waveform, the presence of T21 and defined placental pathological categories. Methods: This case-control study assessed singleton fetuses with karyotypically confirmed trisomy 21 where placental histopathology had been conducted from 2003 to 2015 inclusive,

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Abstracts / Reproductive Toxicology 64 (2016) 29–49

within a university tertiary obstetric centre. This was compared with unselected normal singleton control pregnancies matched within a week of gestation at delivery. Data included birthweight centiles and placental histopathology. Comparisons of Doppler findings across placental pathological categories were performed using statistical analysis. Results: 104 cases were analysed; 52 cases of trisomy 21 and 52 controls. Fetal vascular malperfusion (48.1% vs. 5.8%, p = 0.001) and maturation defects (39.2% vs. 15.7%, p = 0.023) were more common in trisomy 21 placentas. Compared with controls, trisomy 21 fetuses were more likely to have shorter umbilical cords (p = 0.001) and had more UAD abnormalities. Amongst T21 pregnancies, umbilical artery Doppler abnormalities are associated with the presence of maternal vascular malperfusion. Discussion: Fetal vascular malperfusion and maturation defects are more common in trisomy 21 placentas. Abnormal umbilical artery Doppler waveforms are more common in T21 and are associated with maternal vascular malperfusion. Placental disease may explain the increased rate of intrauterine death in T21. http://dx.doi.org/10.1016/j.reprotox.2016.06.104 P-44 Developmental toxicity of differently coated Silver nanoparticles on Xenopus laevis Melissa Saibene 1,∗ , Anita Colombo 1 , Patrizia Bonfanti 1 , Elisa Moschini 1 , Maddalena Collini 2 , Kaja Kasemets 1,2,3 , Paride Mantecca 1 1

Department of Earth and Environmental Sciences, Research Centre POLARIS, University of Milano-Bicocca (Mi), Italy 2 Department of Physic, University of Milano-Bicocca (Mi), Italy 3 Laboratory of Environmental Toxicology, National Institute of Chemical Physics and Biophysics, Tallinn, Estonia

Silver Nanoparticles (AgNPs) are among the most exploited nanomaterials (NMs) in consumer products due to their antimicrobial properties. Size, surface reactivity and coating are critical physicochemical properties responsible for NP toxicity. Surface coatings, often used to functionalize or stabilize AgNPs, can modify surface chemistry or charge, influencing NP toxic profile and biocompatibility. In this study the reproductive and developmental toxicity of positively charged Branched Polyethyleneimine- (BPEI-) and negatively charged Citrate- (Cit-) coated 10nm AgNPs was investigated by Frog Embryo Teratogenic Assay – Xenopus (FETAX). The effects were compared also with the exposure to Silver ions. Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM) analyses were performed to study AgNP physicochemical properties. The results suggest that the positively-charged BPEI-AgNPs are more effective in inducing adverse developmental outcomes. In contrast to Cit-AgNPs, the positively charged BPEI-AgNPs exerted a concentration-dependent effect on lethality and malformations. The BPEI-AgNPs showed the highest teratogenic index, pointing out the role of functional coating in determining the lethal and teratogenic effects. In addiction, Ag+ was strongly embryo-lethal, more than AgNPs. In BPEI-AgNP treated larvae, the histological examination revealed irregular intestinal diverticula coupled with edematous surrounding connective tissue. Small NP aggregates were mapped at level of gut lumen, along enterocyte brush border and inside of enterocytes by two-photon excitation confocal microscopy.

We conclude that a possible teratogenic risk may be associated to BPEI-AgNPs exposure, but the modality of NP-tissue interactions and the teratogenic mechanism will need further scrutiny to be defined. http://dx.doi.org/10.1016/j.reprotox.2016.06.107 P-45 Methods for Conducting Intravenous Juvenile Toxicology Studies in Rats Rose-Marie Gendron 1,∗ , Louise Pouliot 1 , Stephanie Barbeau 1 , Lise Bernier 1 , Elise Lewis 2 , Prägati Coder 3 , Keith Robinson 1 1 Charles River Laboratories, Montreal, Quebec, Canada 2 Charles River Laboratories, Horsham, Pennsylvania, USA 3 Charles River Laboratories, Ashland, Ohio, USA

Rodent juvenile toxicology studies via the intravenous (IV) route may be required for some pharmaceuticals and biopharmaceuticals. Treatments ranging from (slow) bolus to continuous infusion may be used clinically. To allow for the conduct of nonclinical juvenile studies to meet EMA guideline and US FDA guidance similar dose regimens are needed. Appropriate experimental procedures as well as dose volumes and properties of the formulation to be administered must be taken into consideration. Studies were conducted at our laboratories with a range of vehicles that included Dulbecco’s phosphate buffered saline, calcium disodium EDTA, and isotonic sterile saline; administered by daily slow bolus intravenous injection (tail vein) for up to 6 months starting prior to weaning. For IV infusion administration, saline and citrate solutions were used for up to 4 weeks via surgically implanted catheter in the femoral vein (using a jacket/tether set-up) on Day 21 post partum. All animals were continuously infused with 0.9% Sodium Chloride Injection, USP via an in–line 0.22 ␮m PVDF filter until initiation of treatment. A minimal number of repair surgeries were required and the incidence was similar to that seen in adult rats. The animals were 28 days of age at the start of infusion. Additionally, non-surgical methods of slow infusion via the tail vein have been successfully applied in weanling rats at Day 21 post partum, using through-the-needle or over-the-needle catheters. Weekly or twiceweekly infusions (30–60 min each) of saline based solutions have been successfully administered with no adverse consequences. For animals treated by infusion via surgically implanted catheter, there was no mortality and no noteworthy clinical signs; there were slightly lower body weight gains at the start of infusion but had similarly relative body weight gains thereafter, compared to noninfused rats, these differences being attributable, at least in part, to the surgical procedures. Background changes at the infusion sites were noted at necropsy, but were similar to those seen in instrumented adult rats with the same vehicles. IV bolus treatment did not cause any adverse effects in terms of survival, growth or general condition, and local changes at the injection sites were comparable to those seen in adults. In conclusion, it was demonstrated that with appropriate techniques, rats from preweaning ages can be dosed by daily IV injection for up to 26 consecutive weeks. Similarly, rats can successfully be surgically cannulated on Day 21 post partum and be maintained on continuous IV infusion with treatment initiation recommended following a 1 week post-operative period (ie. starting at 28 days of age) for up to 4 weeks in juvenile rats as well as via non-surgical methods of slow infusion via the tail vein. http://dx.doi.org/10.1016/j.reprotox.2016.06.108