Buelke-Sam J

A Developmental Neurotoxicity Evaluation of the Effects of Prenatal Exposure to Fluoxetine in Rats

Fluoxetine is a widely used serotonin reuptake inhibitor effective in the treatment of depression. This experiment assessed the potential developmental neurotoxicity of fluoxetine. Sprague-Dawley CD rats were treated once per day on Days 7-20 of gestation with 0, 1, 5, or 12 mg/kg of fluoxetine (free base) dissolved in distilled water. One control group received water by gavage; animals in this group were provided food and water ad libitum. The second control group (PF) also received water by gavage; animals in this group had their food and water restricted by pair-feeding and watering them to the 12 mg/kg fluoxetine group. Litters were culled to 12 after birth and offspring (male/female pairs) were tested neurobehaviorally at three developmental stages (preweaning, juvenile, and adult). At each stage, two pairs per litter received tests of locomotor activity, acoustic startle, and startle after administration of one of two pharmacological challenges (one pair each receiving fluoxetine or apomorphine). Two pairs were also tested for spontaneous alternation, passive avoidance, and complex learning in a water maze. At the highest dose, fluoxetine caused maternal weight loss during pregnancy, reduced litter sizes at birth, and increased neonatal mortality. No effects on long-term growth or survival were seen. Prenatal fluoxetine exposure produced no significant effects on locomotor activity, spontaneous alternation, passive avoidance, or water maze performance. A few scattered interactions involving treatment group were obtained on startle, but no pattern of treatment-related changes was evident. Regional wet and dry brain weights taken at each stage were not affected by prenatal fluoxetine exposure. The data suggest that fluoxetine is not developmentally neurotoxic in the rat.

The selective estrogen receptor modulator, raloxifene: an overview of nonclinical pharmacology and reproductive and developmental testing.

Raloxifene is a nonsteroidal, selective estrogen receptor modulator being developed by Eli Lilly and Company as a therapeutic agent for postmenopausal osteoporosis. In the ovariectomized (OVX) rat, raloxifene prevents the loss of bone at the distal metaphysis of the femur, proximal tibia, and vertebrae; reduces cancellous bone resorption; and reduces serum cholesterol, but does not cause any significant changes in stromal eosinophilia or uterine epithelium. In estrogen-stimulated OVX rats, raloxifene prevents the morning lowering of serum luteinizing hormone levels, produces a reduction in afternoon serum prolactin levels, antagonizes pituitary weight increase, and antagonizes stimulation of mammary gland development. Raloxifene also has been shown to exhibit antiestrogenic activity in several in vivo and in vitro mammary tumor models. Raloxifene treatment results in regression of endometriosis in both a surgically prepared, rat uterine explant model and in Rhesus macaques diagnosed with spontaneous endometriosis before exposure. Also, uterine leiomyomas in estrogen-stimulated OVX guinea pigs regress after the onset of raloxifene treatment. Raloxifene antagonizes testosterone-induced increases in prostate weight of castrated rats, but does not bind to androgen receptors or affect prostatic 5-alpha-reductase or testicular steroid 17-alpha-hydroxylase activity. A series of preclinical toxicology studies was designed to characterize reproductive and developmental outcomes following various schedules of raloxifene treatment in rats or rabbits. Studies of female reproduction and developmental outcome were conducted primarily at pharmacologic doses (0.1, 1, or 10 mg/kg/d); male reproductive studies used higher doses (10, 30, or 100 mg/kg/d). In this series of studies, male reproductive end points were not affected, whereas embryo implantation, fetal rabbit morphology, and several aspects of offspring development were disrupted by the lowest dose of maternal raloxifene treatment, a profile consistent with estrogen antagonist activity.

The Selective Estrogen Receptor Modulator, Raloxifene: A Segment II/III Delivery Study in Rats

Raloxifene is a nonsteroidal, selective estrogen receptor modulator developed by Eli Lilly and Company as a therapeutic agent for postmenopausal osteoporosis. Raloxifene was administered orally by gavage at doses of 0, 0.1, 1, or 10 mg/kg/d to female CD rats (25/group) on Gestation Day 6 (GD 6) through Postpartum Day 20 (PD 20). Females were allowed to deliver and maintain their progeny until PD 21. All dead pups and pups culled on PD 1 were given internal and external examinations. One pup/sex/litter was assigned to each of the following assessment groups: 1) the primary pair for the F1 generation study, in which survival, growth, development, behavior, indicators of sexual maturation, and reproductive performance were evaluated; 2) terminal necropsy evaluations at PD 21; 3) terminal necropsy evaluations at 60 d of age; and 4) assessments of immune function at 5 to 6 weeks of age. At termination on PD 21, 60, or approximately 140, a necropsy was performed; crown rump and tibia lengths were measured; pituitary weights were taken; and a portion of the anterior pituitary was retained for growth hormone, luteinizing hormone, and prolactin content determinations (control and 10-mg/kg groups only). The remainder of the pituitary and reproductive tissues were retained for histologic evaluations. Dose-related depressions in maternal body weight and food consumption occurred during gestation. Mean gestation length was increased at 1 and 10 mg/kg. Delayed, extended, and/or disrupted parturition occurred in dams given 10 mg/kg, which resulted in a high incidence of maternal morbidity and/or death, increased numbers of dead pups, and the survival of only 66% of live pups to PD 21. Progeny body weights were not decreased at birth, but were depressed progressively in a dose-related manner during the 3-week lactation period. Negative geotaxis and incisor eruption were apparently accelerated in the 1- and 10-mg/kg groups, but eye opening was delayed at 10 mg/kg. Postweaning activity levels, auditory startle, and passive avoidance performance were not affected in the raloxifene groups. Dose-related decreases in spleen cellularity and thymus weights occurred in both sexes, but immune system function, as measured by splenic natural killer cell activity and antibody response to sheep red blood cells, was not affected. Postweaning body weights and growth parameters, as well as pituitary hormone content, were affected in both an age- and sex-specific manner. Preputial separation was not affected, but vaginal patency occurred ca 2 d earlier than controls in females from the 10-mg/kg group. Estrous cycles of the F1 females were not affected during the first two weeks after vaginal opening, but were disrupted at 12 to 14 weeks of age in the 10-mg/kg group. These females showed poorer mating and fertility indices, and litter size was reduced in the two females that were pregnant. Histologically, reproductive organs were not affected in males at any age or in females at PD 21. At PD 60, vaginal mucification occurred in females from the 0.1- and 1-mg/kg groups. At PD 140, the only finding was a high rate of uterine hypoplasia in the 10-mg/kg group, and this finding occurred in the absence of any concomitant ovarian or vaginal changes. These reproductive and developmental findings are consistent with estrogen antagonist activity of raloxifene.

A Reproductive and Developmental Toxicity Study in CD Rats of LY275585, [Lys(B28),Pro(B29)]-Human Insulin

LY275585, [Lys(B28),Pro(B29)]-human insulin, was administered daily by subcutaneous injection at doses of 0, 1, 5, or 20 U/kg. Male rats were treated with LY275585 beginning 2 weeks prior to cohabitation and throughout the mating period. Females assigned to the teratology component of the study were treated for 2 weeks prior to cohabitation with the males and through gestation day (GD) 19. These dams were killed on GD 20 and uterine and fetal examinations were performed. Female rats assigned to the delivery component were treated for 2 weeks prior to cohabitation through postpartum day (PD) 20. Dams were allowed to deliver and maintain their progeny through a 21-day lactation period. After weaning, 1 pup/sex/litter was assigned to the F1 generation, and these animals received no treatment. Survival, growth, behavior, and reproductive performance were evaluated, and reproductive organs were collected for histological evaluation. Treatment of F0 male and female rats with LY275585 resulted in isolated incidences of severe hypoglycemia at 5 and 20 U/kg/day and some modest changes in food consumption and body weight measures at all treatment levels. These changes were anticipated and attributed to the pharmacology of this compound. Mating and fertility of the F0 animals were unaffected by treatment. While slight decreases in fetal body weights and increased fetal runts/litter were observed in the 20-U/kg/day group, PD 1 progeny weights were not affected in the delivery component, and there was no indication of teratogenicity in this study. There were no remarkable treatment-related effects on offspring growth patterns, survival, or reproductive performance, but the F1 animals from the 20-U/kg/day treatment-derived group were more reactive than controls in the startle habituation test. Thus, F0 parental toxicity, related to the hypoglycemic action of LY275585, was found at all doses. A dose of 5 U/kg/day was considered a no-adverse-effect level for developmental toxicity. There were no remarkable effects of LY275585 treatment on F0 or F1 generation reproductive performance at 20 U/kg/day, the highest dose tested in this study.

Developmental toxicity of the dopamine agonist pergolide mesylate in CD-1 mice. I: Gestational exposure☆☆☆

Pergolide was given by oral gavage to mated CD-1 female mice at doses of 0, 1, 20, or 60 mg/kg/day on gestation days (GD) 6-15. Animals assigned to the teratology segment were killed on GD 18 for evaluation of maternal organ weights, and fetal viability, weights and morphology. Animals assigned to the postnatal segment were allowed to deliver and physical development and behavioral performance of the progeny were monitored until weaning. Maternal organ weights were collected at termination after weaning. One F1 offspring per sex per litter was maintained for postweaning physical, behavioral and reproductive assessments and for terminal examinations and organ weight evaluations. No adverse effects of pergolide treatment were found in the 1 mg/kg/day group. Dose-related hyperactivity, chewing and squinting that were consistent with dopaminergic stimulation occurred following dosing in the 20 and 60 mg/kg/day groups; F0 body weights and food consumption were reduced during the initial phase of treatment in the 60 mg/kg/day group. Gravid uterine weights and fetal weights were decreased in the 60 mg/kg/day group of the teratology segment, but there was no indication of teratogenicity in any group. Mammary inflammation, attributed to increased progeny suckling, occurred during the second week postpartum in a few postnatal segment females of the 20 and 60 mg/kg/day groups. Mean negative geotaxis performance was delayed slightly, but mean progeny survival and body weights were not affected. Although after weaning the F1 offspring from the treatment-derived groups tended to weigh more than controls and to perform more effectively in the active avoidance task, these findings were attributed to unusually low values obtained in the control group. Startle amplitudes were increased significantly in the males from the 60 mg/kg/day treatment-derived group. These dose-related maternal and developmental findings were all consistent with the mixed D1/D2 agonist properties of pergolide mesylate, and suggest that only very high doses may result in persistent effects on the developing central dopaminergic systems.

The Selective Estrogen Receptor Modulator, Raloxifene: Reproductive Assessments following Premating Exposure in Female Rats

Raloxifene HCl is a nonsteroidal, selective estrogen receptor modulator developed as a therapeutic agent for postmenopausal osteoporosis. Two studies were conducted that examined the effects of premating exposure to raloxifene HCl. In the first study, adult female CD rats (20/group) were given diets containing 0, 0.01, or 0.1% raloxifene (providing an average of 0, 6, or 63 mg/kg/d, respectively) for 2 weeks, after which the treated diets were replaced with control diet. Following a 2-week period without treatment, each female that had displayed at least three conversions in vaginal cytology from cornified cells to leukocytes was cohabited for 1 to 2 d with an untreated male as she entered proestrus. Females were killed at midgestation and examined for evidence of pregnancy. In the second study, adult female CD rats (40/group) were given oral gavage doses of raloxifene (0, 0.1, 1, or 10 mg/kg/d) for 4 weeks. Immediately or following a 2-week period without treatment, 20 females/group were cohabited with untreated males (1:1) for up to 3 weeks. The females were allowed to deliver and rear their offspring until Postpartum Day 21. Progeny survival, growth, and development were evaluated. Maternal body weight, body weight gain, and food consumption were depressed in all raloxifene treatment groups. Doses > or =1 mg/kg caused disruptions in estrous cycles. In Study 1, 90% of the females treated with raloxifene resumed normal cycling, and fertility was not significantly affected. Although there were no statistically significant differences in time-to-mating, fertility, or liveborn indices in Study 2, females in the 10-mg/kg immediate-cohabitation group had slightly increased gestation lengths and smaller litter sizes. Progeny from these litters were larger on Postpartum Day 1 and had advanced incisor eruption and eye opening. In addition, slight delays were seen in physical landmark appearance in the 0.1- and 1-mg/kg immediate-cohabitation groups and in the 1- and 10-mg/kg delayed-cohabitation groups. Progeny viability, growth, and negative geotactic performance were not adversely affected. In these studies of maternal premating exposure to raloxifene, findings were consistent with established pharmacologic activity of the test chemical. Reproductive effects (disrupted estrous cycles and decreased litter size) occurred at doses > or =1 mg/kg and were generally reversible. Effects on offspring were seen at doses > or =0.1 mg/kg, were of minor importance, and were resolved during the lactation period.

The selective estrogen receptor modulator, raloxifene : Reproductive assessments following preimplantation exposure in mated female rats

Raloxifene is a nonsteroidal, selective estrogen receptor modulator being developed for postmenopausal osteoporosis. As part of an integrated reproductive toxicity assessment, two studies were conducted in which raloxifene was administered orally to CD rats during Gestation Days (GD) 0 through 5. In each study, animals received daily raloxifene doses of 0, 0.1, 1, or 10 mg/kg. In Study 1, GD 20 evaluations of maternal reproductive parameters identified dose-related increases in pre- and postimplantation loss, reductions in the numbers of corpora lutea and live conceptuses, and reduced fetal weight. The low fetal weights were consistent with an extent of morphologic development that corresponded to developmental ages up to 8 d younger than GD 20. Study 2 characterized the potential impact of this disrupted and apparently delayed implantation on gestation length, parturition, and progeny viability. Dams were allowed to deliver and rear their offspring through Postpartum Day 21. Gestation lengths were extended up to 1 week, and litter sizes were reduced in a dose-dependent manner. Nevertheless, parturition occurred normally and pup morphology, survival, and physical and behavioral development were unaffected.

Developmental toxicity of the dopamine agonist pergolide mesylate in CD-1 mice. II: Perinatal and postnatal exposure☆

Pergolide mesylate is a dopamine agonist and, therefore, reduces prolactin secretion. In Experiment I, pregnant mice were given oral doses of 0, 0.1, 0.3, 1.0 or 3.0 mg/kg/day pergolide on GD 15 through PD 10 or 20 to identify a tolerated dose which would not markedly reduce offspring survival during late gestational and lactational exposure. Offspring survival was not affected at any dose, but dose-related decreases in progeny body weights occurred at weaning. On PD 10, suckling-induced increases in maternal serum prolactin concentrations did not occur in dams treated with 3.0 mg/kg/day. In Experiment II, pregnant mice were given oral doses of 0, 0.002, 0.1 or 3.0 mg/kg/day pergolide on GD 15 through PD 20. Dams were allowed to deliver and maintain their offspring throughout a 21-day lactation period. Growth and behavioral performance of one F1 male and one F1 female per litter were monitored, followed by a reproduction trial and terminal organ weight measurements. There were no treatment-related effects on maternal body weights, food consumption, or terminal organ weights and pathology. Three dams showed overt signs of mammary inflammation and lactational insufficiency and mean progeny survival was decreased slightly in the 3.0 mg/kg/day group. There were no adverse effects on growth, development or reproductive performance in the F1 treatment-derived generation. Neonatal negative geotaxis, 1-h activity levels at 30 and 60 days of age, auditory startle habituation at 55 days of age, and two-way active avoidance performance at 65 days of age were not affected significantly by treatment. Thus doses of pergolide that did not inhibit lactation completely in the F0 dams were found to have no enduring effects on offspring development.

The use of repeated measures analyses in developmental toxicology studies.

In many toxicology studies, it is common to take the same measurements on an individual animal at several time points (e.g., body weight across days or weeks, activity levels either within a single test session or across days). Such repeated measures for both maternal and offspring endpoints routinely are incorporated into developmental toxicology studies to allow characterization of the profile of treatment-related effects over time. To permit a valid statistical analysis, repeated measures studies are characterized by: random assignment of treatment to experimental units, measurement of the variable(s) of interest at the same time points for all animals, and avoidance of systematic overlaying of treatment and potential response gradients. Assumptions for and advantages of the use of a repeated measures analysis of variance versus single-factor analyses at each time point are illustrated in two data sets from a rat perinatal and postnatal study. The variance-covariance structure of repeated measures designs dictates that adjustments are necessary to provide protection against inflated Type 1 error rates. Communication between statisticians and toxicologists which allow the implementation of such analyses can improve the interpretation of data resulting from repeated measures study designs.