Excretion profile of DI-(2-ethylhexyl) phthalate (DEHP)in pregnant and non-pregnant female rats and mice

PosterSession4C. Reproductive Toxicology with TOX or Tz showed synergistic effects on CNS malformations, The results suggest that there is a site-specific and dose-related interactive dysmorphogenesis elicited by TOX or its congeners and high levelsof glucose in rat embryonic development.

IP4C39 I ELIMINATION OF METHOXYETHANOL AND METHOXY

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Sprague Dawley rat and NewZealand White rabbit routinely used in reproductive toxicologystudies in our laboratory, closure of the hard palate is not complete in all fetuses until days 17and 19 of pregnancy respectively. We recommend,therefore, an exposureperiod from day 6 (for both species) to days 17 and 19 of pregnancy inclusive for embryotoxicity studies in the rat and rabbit respectively in order to satisfy the ICH requirements.

ACETIC ACID IN MALE AND FEMALERATS

L. Aasmoe *, M. Mathisen,G. Sager, J. Aarbakke, Dept. of Clinical Pharmacology, University Hospital of Tromse, Norway The glycolether methoxyethanol (ME) is used as a solvent, and has teratogenic, spennatotoxic and hematotoxic effects. This glycolether is oxidized to the corresponding alkoxyacetic acid; methoxy acetic acid (MAA), by alcohol dehydrogenase. This metabolic conversion of the glycolether is a prerequisite for development of toxicity, as the toxic effects have been shown to be due to the alkoxyacetic acid metabolite. Alcohol dehydrogenaseappears to be one of several enzymes that displays sexually dimorphic activities in adult rats, and is probably at least in part under the control of testosterone. In experimental studies it has been shown that the activity level of alcohol dehydrogenase in rat liver is strongly sex-dependent, with higher activities in females than in males. The elimination of ME and its toxic metabolite MAA was studied in male and female rats. The rate of ME-elimination after i.p, injection of 150 mglkg ME was significantly higher in females compared to males. The elimination half-life was estimated to 49 ± 10 min in male rats and 28 ± 5 min in female rats. There was no gender difference in the elimination of MAA after i.p. injection of 100 mglkg ME. The elimination of MAA was markedly slower compared to ME, and was estimated to 12.6 ± 1.3 hours in males and 14.1 ± 1.4 hours in females. Accumulation of the toxic metabolite MAA following frequent exposures to ME can thus occur, and it remains to be determined whether there is a sex-difference in the susceptibility to toxic effects following exposure to ME.

IP4C40 I

DAYOF CLOSURE OF THE FETALHARDPALATE IN THE SO RATAND NZW RABBIT

E.K.S. Marsden *, F. Roche, P.C. Barrow. Chrysalis Preclinical Services - Europe, Les Oncins, BP 118, 69593, L'Arbresle, France The internationally harmonised reproductive study design, ICH 4.1.3., requires exposure of females during pregnancy from implantation through to closure of the fetal hard palate. It is therefore necessary to determine the day of closure of the palate to demonstrate that a testing protocol mcets the regulatory requirements. Fifteen Sprague Dawley rats (strain leo OFA.SD (lOPS Caw» and twelve New Zealand White rabbits (Strain NZW I.N.R.A. A 9077) were obtained from the suppliers on day 0 of presumed pregnancy. They were housed and maintainedunder our standardenvironmental conditions and selected females were necropsied on each of days 15. 16 and 17 for the rat, and 17. 18 and 19 for the rabbit. post coitum. A total of 54, 48 and 38 rat fetuses, and 10, 51 and 43 rabbit fetuses were availablefor examinationon each of the specified days respectively. Following external examination, each fetus was killed and then examined for closure of the hard palate following fixation; the palate was categorised as open. partly closed or closed. For the rat, all fetuses had open palates on day 15 of pregnancy. On day 16, closure was complete in 13/48 fetuses, 29/48 had partial closure, and the remaining 6 had open palates. On day 17, closure was complete in all fetuses. For the rabbit, 8110 fetuses had open palates on day 17 and tIie remaining 2 had partial closure. On day 18, closure was complete in 17/51 fetuses, 31151 had partial closure and the remaining 3 had an open palate. On day 19. closure was complete in all fetuses. The results indicate that for the strain of

IP4C41 I OPC-14523: RECEPTOR BINDING CHARACTERIZATION STUDIES ON A NOVEL ANTIDEPRESSANT AGENT

O.M. Adeyemo *1, N.J. Browder!, K. Tottori2 • T. Kikuchi2 . 2Tokushima InstituteofNew Drug Research, Otsuka Pharmaceutical Corporation, Tokushima; Japan; JOtsukaAmerica Pharmaceutical, Inc. Rockville, Maryland, USA OPC-14523, a quinolinone derivative, is a novel psychoactive drug in development for use as an oral antidepressant agent. It is characterized by fast onset of action and minimal toxicity. It exhibits sigma receptor agonism, serotonergic HTlA receptor agonism, and serotonin (5-HT) reuptake inhibition. Receptor binding characteristics in the rat and guinea pig brain showed that OPC-14523 has affinity for sigma receptors (ICso = 128 oM), similar to that of selective sigma receptor agonists (l,3-di-o-tolyl-guanidine (DTG) and (+)-pentazocine) at ICso values of 267 oM and 147 oM, respectively. OPC-14523-induced improvementin the Consciousness Disturbance Mice Model was antagonized by rimcazole, a selective sigma receptor antagonist. Binding of OPC-14523 to 5-HTIA receptors in the rat brain exhibited high affinity with an IC50 = 2.3 nM compared to that of other selective 5-HTlA receptor agonists (i.e. serotonin with an 1C50 of 3.8 oM and 8-hydroxy-2-(di-n-propylamino)tetralin with an ICso of 4.1 oM). OPC-14523 dose-dependently decreased serotonin biosynthesis (5-hydroxytryptophan content) in the mouse forebrain in the presence of an aromatic L-amino acid decarboxylase inhibitor. These changes are related to its effects on presynaptic and postsynaptic 5-HTIA receptors and its inhibitory effect on serotonin reuptake, Similarly, serotonin reuptake ex vivo data in the rat synaptosomes indicated that OPC-14523 inhibited serotonin reuptake in a concentration-dependent manner with an ICso value of 26.9 oM, a value that is comparable to that of f1uoxetine and superior to that of imipramineand trazodone.

IP4C42 I

EXCRETION PROFILE OF DI-(2-ETHYLHEXYL) PHTHALATE (DEHP)IN PREGNANT AND NON-PREGNANT FEMALERATSAND MICE

C. Nativelle" , K. Picard", M.e. Chagnon", J.C. Lhuguenot", J.F. Regnier *2. J ENSBANA, laboratoire de Toxicologie Alimentaire, F-2IOOO Dijon; 2 Elf-Atochem SA, Departement de Toxicologie Industrielle, F-92091 Paris-ill-Defense, France Di-(2-ethylhexyl) phthalate is extensively used as plasticiser for polyvinyl chloride. Developmental toxicity studies in rodents have shown that mice were more sensitive than rats to the DEHP-induced embryotoxicity and foetal malformations. Metabolic profileof DEHP has been essentially established for rats, and little information is available on mice, and on the influence of pregnancy. In this study, pregnant and non pregnant Wistar rats and CD-I mice were fed by gavage with either a single dose of 1000 mg unlabelled DEHPlkg, or the repeated oral administration of 200 or 1000 mg [ring)4Cj-DEHPlkg for 10 days to non-pregnant animals, and from day 5 to day 16 of gestation. Urine and faeces were collected daily, and DEHP and its metabolites were identified and quantified by GC·MS. DEHP and its metabolites were mainly eliminated in urine and faeces within 24 hours and completely after 72 hours in both pregnant and non-pregnantrats and mice. Mono-(2-ethylhexyI)

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Poster Session 4C. Reproductive Toxicology

phthalate is the major metabolite in mice urine but is absent in rat urine. The other major DEHP metabolites (metabolites I and V from w-oxidation and ,a-oxidation and metabolites VI and IX from w-l oxidation) were mainly excreted via the urine of both species, and no qualitative differences were observed between pregnant and nonpregnant animals. In rats, the major metabolites are VI and IX. Phase II metabolites were also identified in mouse urine, nevertheless the activity glucuronide pathway decreased over the treatment. In both species, the metabolism rate seems slightly greater in pregnant than in non-pregnant animals. In all cases, DEHP was identified in faeces and was absent in urine, suggesting that a large part of DEHP was not absorbed. In conclusion, the results suggest that the difference in species-sensitivity to the developmental toxicity could be related with differences in metabolism.

I P4C43!

THE ENHANCEMENT OF DIABETES TERATOGENICITY BY MATERNAL RESTRAINT STRESSIN THE MOUSE

M. Salazar *, D. Hernandez, G. Brenes, S. Salazar. Departamento de Toxicologia; Escuela Nacional de Ciencias Biologicas, lnstituto Politecnico Nacional, Mexico D.F., Mexico Diabetes and maternal stress, respectively, is known to induce several developmental toxicity in experimental animals. These factors, in combination with known teratogenic agents has been shown to increase the teratogenicity of such agents. It has been well established that diabetic pregnant women may be exposed to various types of stress in different places. Thus it is of interest to determine whether simultaneously exposing pregnant females to diabetes and stress, enhances, the production of toxic effects in the developing offspring. Six treatment groups were used: control (C), diabetes (D), food/water-deprived (FWD), restraint only (R), diabetes + food/water deprive (D + FWD) and diabetes + restraint (D + R). Diabetes was induced by administration of streptozotocin on day 7 of gestation and restriction was applied during 12 h on day 9, by supine position of animals. FWD group mice were deprived of food and water during the same time period. All animals were sacrificed on day 19 of gestation and fetuses were subjected to teratological evaluation. The results show that maternal stress strengthened the embryotoxicity of diabetes which was reflected in embryonic mortality, decreasing degree of ossification, as well as in a qualitative increase in the severity of the skeletal malformations. It is suggested that stress during pregnancy, combined with uncontrolled diabetes, may result in an enhancement of prenatal toxicity to the progeny. Such results may aid our understanding of the role that maternally mediated effects can have in affecting developmental outcome.

IP4C44 I PRENATAL INTERACTIVE EFFECTS OF HYPERTHERMIA AND DIABETES ON MOUSE FETUS

G. Chamorro *, G. Brenes, D. Hernandez. Departamento de Toxicologic, Escuela Nacional de Ciencias Biologicas, lnstituto Politecnico Nacional, Mexico D.F., Mexico Hyperthermia appears to be capable of causing congenital defects in all species and may act alone or synergistically with some chemicals. On the other hand, experiments in rodents systems have demonstrated also a higher incidence of morphological abnormalities in embryos obtained from diabetic pregnancies (l). In the present study, we have investigated whether the diabetic prenatal state enhances the teratogenicity of hyperthermia in mice. Pregnant mice were divided into eleven groups: diabetic (D), hyperthermia 39°C (H39), 40°C (H40), 41°C (H4l), 42°C (H42), 43°C H43), D + H39, D + H40, D + H41, D + H42, and D + H43. Diabetes was induced by administration of streptozotozin on day 7 of gestation, and hyperthermia, on day 8 for 10 minutes, by partial immersion of animals in water at the

desired temperature. Another group of mice treated at 37°C was used as control. All animals were killed on day 19 of gestation and fetuses were examined for external and internal abnormalities. Animals of groups D + H41-D + H43, lost significantly more weight than the concurrent controls. Significant increases in embryo/fetal mortality were observed in the offspring of groups D + H40 and D + H43, although in D + H41 a protective effect was observed. Incidences of visceral malforma-tions were found in D + H41 and D + H42, but no in D + H43. It is concluded that hyperthermia potentiates the teratogenic effects of maternal diabetes, although a thermotolerance phenomen can be produced to some temperatures. [1] Lin, Y. et al., (1995). Teratogen. Carcin. Mut. 15: 147-153.

HISTOLOGICAL ALTERATIONS IN THE XENOPUS IP4C45 I LARVAE EXPOSED TO 2,3,7,8-TETRACHLORODIBENZo-P-DIOXIN (TeDD) AT EMBRYONIC AND EARLY LARVAL STAGES

Shin Mima *, Michiko Sakamoto, Takashi Tanimura. Department of Anatomy, Kinki University School ofMedicine, Osaka, Japan We previously reported that TCDD causes death, edema and the growth retardation in Xenopus larvae. The present study was to examine histological alterations of Xenopus larvae at 12 days after fertilization. Xenopus embryos were continuously exposed to 200 ppb of TCDD for 5 days from day 0-5 after fertilization. The larvae were histologically examined using 10 uii: paraffin and I {tm Epon sections. In the TCDD group, contents in esophagus, stomach and intestine were markedly reduced. Edema was noted in the ocular region, in the subcutaneous layer, between the muscular layer and serosa of thoracoabdominal wall, and in the submucous layer of digestive organs. The ascites in abdomen were also observed. Submucosal vessels were markedly dilated in the digestive tract. Various degrees of degenerative epithelia were often observed in the lumen of the digestive tract, particularly numerous in the duodenum. The disturbance of the reticular structure of hepatic parenchymal cells and sinusoidal capillaries was observed in the exposed group. The results suggest that the histological alterations in the digestive and circulatory systems induced by TCDD are one of the causes of edema formation and death of the exposed larvae.

I P4C46 I DEVELOPMENTAL TOXICITY EVALUATION OF N-METHYLDIETHANOLAMINE (MDEA) APPLIED CUTANEOUSLY TO CD® RATS

H.-W. Leung *, B. Ballantyne. Applied Toxicology Group, Union Carbide Corporation, Danbury, CT, USA Aqueous N-methyldiethanolamine (MDEA) was applied cutaneously (6 hr/day) to pregnant CD® rats from gestation day (gd) 6-15, inclusive. Doses employed were 0, 250, 500 and 1000 mglkg/day and were selected on the basis of toxicity responses determined from range finding studies on pregnant rats. Monitors for maternal toxicity included body weight and food consumption measurements, and clinical observations including skin irritation scores. Prior to scheduled necropsy on gd 21, blood was obtained from dams for hematologic measurements. At necropsy, liver and kidney, which are putative target organs, were weighed. Fetuses were evaluated for body weight and for external, visceral and skeletal anomalies. Severe skin irritation, characterized by necrosis, ecchymoses, erythema, edema, exfoliation, crusting and excoriation, were noted in dams receiving 1000 tug/kg/day during the dosing period and persisted for several days subsequent to treatment. However, by gd 21, substantial repair of the dosing site had occurred. Less severe skin irritation was noted at 500 rug/kg/day, There were no effects of treatment on maternal body weight, gestational weight gain or food consumption. Maternal liver or kidney weights were also unaffected. Mild anemia