24 Poster abstracts / Reproductive Toxicology 41 (2013) 21–34

Teratogenic effects of mixtures of azole fungicides assessedin vitro and in vivo: II – Binary mixtures with increasing levelsof one of the two components

Francesca Di Renzo 1,∗, Angelo Moretto 2,3, Elena Menegola 1

1 Università degli Studi di Milano, Department of Biosciences, Milano,Italy2 International Centre for Pesticides and Health Risk Prevention (ICPS)University Hospital Luigi Sacco, Milano, Italy3 Università degli Studi di Milano, Department of Occupational andEnvironmental Health, Milano, Italy

On the basis of results obtained by co-exposure to “similarpotency levels” of the agrochemical fungicides triadimefon (FON)and flusilazole (FLUSI), and to better characterize the contribu-tion of each component to the observed effects, with the presentwork the in vitro and in vivo teratogenicity of co-exposures tovariable combinations of compounds has been evaluated. Embryoswere exposed to the No Observed Effect Level (NOEL) of one azole(“fixed”) and increasing levels of the other selected azole (“mov-ing”). The final aim was to compare the dose–response curve of the“moving” molecule alone or in the presence of a non-effective levelof a molecule with a suspected same mode of action.

In vitro protocol: E9.5 CD rat embryos were cultured for48 h in presence of solvent alone (ethanol) or in presence ofFON (6.25–50 �M), FLUSI (1.56–12.5 �M) or mixtures of FON(6.25 �M) + FLUSI (1.56–3.125–6.25 �M) or FLUSI (1.56 �M) + FON(6.25–12.5–25 �M). At the end of the culture period, embryoswere morphologically examined. A clear concentration–responsewas observed both for the single fungicides and for mixtures.The observed malformations were specifically at the level of thebranchial apparatus (reduction and fusion of branchial arches). Theco-exposure caused a shift of concentration–response curve in bothcases.

In vivo protocol: E8 CD1 mouse females were treated bygavage with the solvent alone (acetone:corn oil 1:9) or in pres-ence of FON (75–300 mg/kg), FLUSI (37.5 mg/kg) or mixtures ofFLUSI (37.5 mg/kg) + FON (75–225 mg/kg). Fetuses were examinedat term of gestation for external abnormalities. The specific mal-formation observed at term of gestation was palatoschisis. A clearconcentration–response was observed both for the single fungi-cides and for mixtures.

The data obtained after in vitro and in vivo exposure sug-gest that the basal low level of one azole residue could shift thedose–response curve of the “moving” fungicide, supporting theadditive effects of the co-exposure to azole fungicides for mam-malian developing embryos. Our results, therefore, suggest thatazoles causing this effect should be grouped together for risk assess-ment.

http://dx.doi.org/10.1016/j.reprotox.2013.06.039

Relevance of kinetics and histopathology as part of the embry-onic zebrafish screening test

André Wolterbeek, Vera van der Weijden, CorSnel, Anna Beker,Didima de Groot, Aswin Menke

TNO, Zeist, The Netherlands

Growing awareness to apply the principles of Replacement,Refinement and Reduction (3Rs) of animals in regulatory testingdrives the need for alternatives identifying potential toxic agentswith accuracy, speed, reliability and respect for animal welfare.So far, for complex endpoints like reproduction and develop-mental toxicology, animal-free in vitro models are limited andcover only a restricted part of the reproductive cycle. Various

characteristics warrant Zebrafish (embryos and/or larvae) an idealnon-mammalian whole organism model that could bridge gapsbetween in vitro cell systems and complex reproduction studiesin mammals, e.g. small size, ease of obtaining high number ofprogeny, external fertilization, transparency and rapid develop-ment of embryo, and a basic understanding of its gene functionand physiology.

Our research shows that morphological examination ofzebrafish embryos/larvae, predicts toxicity of chemical and phar-maceutical agents with high certainty and proves to form a reliabletotal organism approach to study embryo- and developmental(neuro)toxicity. A full histopathological survey of the embryo’scombined with high-end analytical methods appears to furtherincrease the applicability domain of the zebrafish in toxicityscreening studies. Further refinement with respect to the physicaland chemical properties of test compounds determining the bodyburden and tissue distribution, increases the predictability of theassay.

http://dx.doi.org/10.1016/j.reprotox.2013.06.040

Renal and skeletal hypoplasia in mouse fetuses following mater-nal exposure to aflatoxin B1 during gestation

Yousef M. Abdulrazzaq 1, Rengasamy Padmanabhan 2, Salim A.Bastaki 3, Jose Kochiyil 1, Mohamed Shafiullah 3

1 Department of Paediatrics, Faculty of Medicine and Health Sciences,UAE University, PO Box 17666, Al Ain, United Arab Emirates2 Division of Basic Medical Sciences, School of Medicine, Mercer Uni-versity, Macon, GA, USA3 Department of Pharmacology, Faculty of Medicine and Health Sci-ences, UAE University, PO Box 17666, Al Ain, United Arab Emirates

There is a paucity of information on the reproductive toxiceffects of aflatoxins and the data in the literature are largely basedon poorly described methodology. Our aim was to evaluate theconsequences of aflatoxin B1 (AFB1) administered during earlyand late stages of organogenesis on fetal growth and developmentin the mouse. We administered groups of mice a single dose of20 mg/kg AFB1 dissolved in DMSO either intraperitoneally (IP) ororally on gestation days (GD) 7, or 13. The controls received a pro-portionate volume of DMSO. Fetuses were collected on GD 18. Thistreatment was found to be maternally non-toxic. However AFB1-treatment resulted in significantly increased incidence of fetuseswith intrauterine growth restriction (IUGR) weighing 2 or morestandard deviations (SD) less than the mean of the controls. A sig-nificant number of fetuses of AFB1-treated mice also were foundto have developed hypoplastic kidneys. The AFB1 group fetusesalso had delay in ossification and maturity of the supraoccipitalbone development, cervical and coccygeal vertebral bodies, and thebones of the fore and hind paws. Another major finding was theincreased incidence of minor malformations, such as the presenceof cervical ribs and sternal anomalies. The results of this study showthat the late organogenesis stage of development is particularlysusceptible to the deleterious effects AFB1 in mice.

Keywords: Aflatoxin; Mouse; Fetal skeletal hypoplasia;Intrauterine growth restriction; Hypoplastic kidneys.

http://dx.doi.org/10.1016/j.reprotox.2013.06.041