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.

The EpiOcular™ Eye Irritation Test is the Method of Choice for the In Vitro Eye Irritation Testing of Agrochemical Formulations: Correlation Analysis of EpiOcular Eye Irritation Test and BCOP Test Data According to the UN GHS, US EPA and Brazil ANVISA Classification Schemes.

The Bovine Corneal Opacity and Permeability (BCOP) test is commonly used for the identification of severe ocular irritants (GHS Category 1), but it is not recommended for the identification of ocular irritants (GHS Category 2). The incorporation of human reconstructed tissue model-based tests into a tiered test strategy to identify ocular non-irritants and replace the Draize rabbit eye irritation test has been suggested (OECD TG 405). The value of the EpiOcular™ Eye Irritation Test (EIT) for the prediction of ocular non-irritants (GHS No Category) has been demonstrated, and an OECD Test Guideline (TG) was drafted in 2014. The purpose of this study was to evaluate whether the BCOP test, in conjunction with corneal histopathology (as suggested for the evaluation of the depth of the injury( and/or the EpiOcular-EIT, could be used to predict the eye irritation potential of agrochemical formulations according to the UN GHS, US EPA and Brazil ANVISA classification schemes. We have assessed opacity, permeability and histopathology in the BCOP assay, and relative tissue viability in the EpiOcular-EIT, for 97 agrochemical formulations with available in vivo eye irritation data. By using the OECD TG 437 protocol for liquids, the BCOP test did not result in sufficient correct predictions of severe ocular irritants for any of the three classification schemes. The lack of sensitivity could be improved somewhat by the inclusion of corneal histopathology, but the relative viability in the EpiOcular-EIT clearly outperformed the BCOP test for all three classification schemes. The predictive capacity of the EpiOcular-EIT for ocular non-irritants (UN GHS No Category) for the 97 agrochemical formulations tested (91% sensitivity, 72% specificity and 82% accuracy for UN GHS classification) was comparable to that obtained in the formal validation exercise underlying the OECD draft TG. We therefore conclude that the EpiOcular-EIT is currently the best in vitro method for the prediction of the eye irritation potential of liquid agrochemical formulations.

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.

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).