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Background We have previously reported that exposure of embryos to 13-cis-retinoic acid (cRA) results in an abnormal phenotype of the fetal cerebellum. In this study, we analyzed early changes in the cerebellar anlagen (midbrain–hindbrain junction) as well as lesions of the fetal cerebellar vermis after a teratogenic dosing regimen of cRA in the macaque model. Methods We examined embryo coronal sections of the midbrain–hindbrain junction immunostained for Pax-2, Engrailed-2 (En-2) and Krox-20. To characterize the cerebellum foliation and fissure formation processes, we analyzed vermal cortical cell layer development and the number and depth of the major fissures on sagittal sections of fetal vermis. We also examined Purkinje cell development in vermal sections immunostained for CD3. Results Compared with controls, there was a consistent truncation of the midbrain–hindbrain region of early embryos exposed to cRA. The cRA-induced fetal vermis lesions included inhibition in its anteroposterior growth, altered folial patterning, a general loss of prominence of the fissures accompanied by a total loss of sublobular fissures, and changes in cortical cell layer development. CD3+ Purkinje cells were abnormally dispersed deep into the molecular layer in the vermis. Conclusions Our findings indicate that the effects of cRA on the developing cerebellum involve interference with the hierarchy of complex cellular and genetic interactions that lead to the growth and subdivision of the cerebellum into smaller units. The regional vermal defects may be related to early postnatal functional deficits. Teratology 63:65–76, 2001. © 2001 Wiley-Liss, Inc.

Background: The 39-year history of the Teratology Society is reviewed. An abbreviated history is outlined in table form, along with listings of the Warkany Lectures, the postgraduate courses, and officers of the Society. Methods: A year-by-year description of the events, including the scientific and social content of the annual meetings and changes in the business of the Society, is given, in many cases using comments from the past presidents. The valuable and unique diversity of the members is discussed and illustrated, presenting the disciplines and main research area of the presidents. The number of submitted abstracts and the various categories are tabulated, averaging the number and type over four periods. Within the past 10 years, a significant increase in the number of abstracts dealing with epidemiology and developmental biology is evident. The Society’s development is compared with that of a human, and the question is asked: Have we reached the maturational stage of old age or senescence, or is the Society still maturing gracefully? This question needs further discussion by all the members. Results: During the past 40 years, we have developed the scientific basis to prevent birth defects caused by rubella, alcoholism, and folate deficiency, as well as many other prenatal exposures. Conclusions: We must now engage in the political battles to obtain the resources needed to conduct further research and to implement the prevention programs, as well as to provide care and rehabilitation for persons with birth defects.

13-cis retinoic acid (RA) is a causative agent for human/monkey retinoic acid embryopathy (RAE), in which the most common type of malformation is microtia or anotia. In the present study, malformed ears of monkey fetuses exposed to RA during early embryogenesis were analyzed and revealed a subtype of defects., i.e., apparent duplication of the external/middle ear. A part of the posterior auricle appeared to be ectopically formed in the anterior auricular region or in the region posterior to the auricle. Additionally, there was duplication of the zygomatic arch, malleus, and incus. In order to characterize possible pathogenetic events underlying these malformations, embryos at selected stages were collected after dosing dams with RA at 5 mg/kg/day during gestational days 12-27. Cellular retinoic acid binding protein I whole-mount immunostaining showed that RA induced specific alterations in the migration of cranial neural crest cells (NCC). NCC en route to the second pharyngeal arch were bifurcated, and some of these NCC migrated abnormally into the first and/or third arches, which may underlie external ear duplication. Scanning electron microscopy and neurofilament immunostaining provided evidence that there was partial duplication of trigeminal nerve/ganglion following RA insult. The duplication of NCC neuronal derivatives in the first pharyngeal arch is consistent with duplication of NCC mesenchymal components (zygomatic arch, malleus, and incus). Therefore, RA-induced alterations in cranial NCC migration patterns are likely to be a pathogenetic event underlying ear malformations (including duplication) of RAE in monkeys.

In genetic male fetuses, dihydrotestosterone (DHT) plays an important role in normal prostatic and external genital differentiation. The enzyme steroid 5-alpha reductase (5 alpha R) catalyzes the conversion of testosterone (T) to DHT. The importance of 5 alpha R in sexual differentiation is evident from the study of human genetic males who congenitally lack this enzyme and consequently develop ambiguous genitalia. These individuals are specifically deficient in the type 2 isozyme, whereas the normal type 1 isozyme activity has been found. The purpose of this study was to determine 1) the suitability of the rhesus monkey for testing the safety of 5 alpha R inhibitors when administered during pregnancy and 2) the potential risk of administering a known type 2 5 alpha R inhibitor, finasteride, during the critical period of internal and external genital differentiation in rhesus monkeys. In vitro studies were also performed on selected rhesus monkey tissues to determine the distribution of the 5 alpha R isozymes. Gravid monkeys were treated once daily from gestational days (GD) 20 to 100. Sonographic monitoring was performed during the course of gestation to monitor viability, growth, and organ system development. Detailed fetal evaluations for developmental abnormalities were performed at term (GD 152 +/- 2). A group of 13 pregnant monkeys ("positive control") were given a high oral dose (2 mg/kg/day) of finasteride to demonstrate that inhibiting type 2 5 alpha R results in specific external genital abnormalities in male fetuses. Thirty-two pregnant monkeys were administered an intravenous (i.v.) formulation of finasteride at doses of 8, 80, or 800 ng/day. The highest i.v. dose selected was at least 60-750 times the semen levels of finasteride in man given orally 5 or 1 mg/day, respectively. Seventeen vehicle-control pregnant monkeys were also included. Administration of a high oral dose (2 mg/kg/day) of finasteride resulted in external genital abnormalities characterized by hypospadias, preputial adhesions to the glans, a small underdeveloped scrotum, a small penis, and a prominent midline raphe in male fetuses; however, no developmental abnormalities were seen in female fetuses. Similarly, no abnormalities were observed in either male or female fetuses of mothers given iv doses (8, 80, or 800 ng/day) of finasteride during pregnancy. The in utero sonographic findings in fetuses correlated with the gross findings at term. These studies have shown that external genital abnormalities can be produced in male monkey fetuses when exposed to a high oral dose (2 mg/kg/day) of finasteride, whereas no abnormalities were observed in fetuses exposed to the i.v. formulation of finasteride. Detailed in vitro studies demonstrated that the rhesus monkey also has two 5 alpha R isozymes (types 1 and 2) with a tissue distribution similar to that seen in man and, furthermore, that finasteride is a potent, mechanism-based inhibitor with selectivity for both human and rhesus type 2 5 alpha R. These studies have demonstrated that the monkey is a suitable model for assessing the safety of 5 alpha R inhibitors administered during pregnancy.

Retinoids often exhibit a complex metabolic pattern and differential transplacental kinetics, which make it difficult to pinpoint the proximate compound responsible for the observed teratogenic effect. We have therefore studied the pharmacokinetics and metabolism of all-trans-retinoic acid (all-trans-RA) in cynomolgus monkeys following application of a nonteratogenic dosing regimen and compared the results with corresponding data from a previous study with a teratogenic dosing regimen with 13-cis-RA [Hummler et al. (1994) Teratology 50:184-193]. All-trans-RA was administered to pregnant cynomolgus monkeys (Macaca fascicularis) by nasogastric intubation at a dose of 5 mg/kg body wt once daily from gestational day (GD) 16 to 26 and twice daily at 8-h intervals from GD 27 to 31. Examination of the fetuses of four dams on GD 100 +/- 2 showed no embryotoxic or teratogenic effects of the applied dosing regimen (Experiment 1). Maternal plasma retinoid pharmacokinetics on GD 16, 26, and 31 as well as embryonic retinoid profiles after the last drug administration on GD 31 were determined in thirteen further dams (Experiment 2). All-trans-RA reached much lower plasma concentrations after the last two treatments on GD 31 than after the first one on GD 16 and the eleventh one on GD 26 (0-24-h area-under-the-concentration-time-curve (AUC) values: 104 +/- 59 ng x h/ml (after the last treatment on GD 31), 189 +/- 110 (GD 16) and 393 +/- 305 ng x h/ml (GD 26). The predominant plasma metabolites of all-trans-RA were its beta-glucuronide and the beta-glucuronide of all-trans-4-oxo-RA. Both of these retinoids accumulated in the plasma during the period of treatment and displayed AUC values 5- to 30-fold higher than those of all-trans-RA. Embryonic concentrations of all-trans-RA were not increased over endogenous levels after the last administration on GD 31 when plasma concentrations were low. To evaluate the placental transport of all-trans-RA in the presence of high plasma concentrations, a further experiment was performed, in which a single dose of all-trans-RA (10 mg/kg body wt) was given to four pregnant monkeys on GD 31, and plasma pharmacokinetics as well as embryonic concentrations of retinoids at 4 h post-treatment were determined (Experiment 3). This dosing schedule yielded high plasma concentrations of all-trans-RA, while embryonic concentrations were about 40% of plasma levels. Based on the plasma AUC values on GDs 16 and 26 obtained in Experiment 2 and the degree of placental transfer, as determined on GD 31 in the presence of high plasma levels in Experiment 3, we estimated embryonic AUC values for the 24-h period following the nonteratogenic doses on GDs 16 and 26 in Experiment 2. These AUC values were similarly high to the embryonic AUC value of all-trans-RA obtained after application of the teratogenic dosing regimen with 13-cis-RA [Hummler et al. (1994) Teratology 50:184-193]. In addition, plasma AUC values of all-trans-RA were 2- to 7-fold higher after all-trans-RA administration (present study) than after dosing with the teratogenic dose of 13-cis-RA. These results strengthen our recent suggestion that the teratogenic effects induced in cynomolgus monkeys by 13-cis-RA treatment cannot solely result from the action of all-trans-RA, but may involve 13-cis-RA and 13-cis-4-oxo-RA, which could act directly or function as transport vehicle.

Parke-Davis Pharmaceutical Research, Ann Arbor, Michigan 48105 (J.WH.); Miles, Inc., Elkhart, Indiana 46515 (K.G.H.); USFDA, Rockville, Maryland 20852 (M.A.S., J.A.C.); California Regional Primate Research Center, Davis, California 95616 (A.G.H.); Hoffman-LaRoche, Inc., Nutley, New Jersey 07110 (N.D.A.); Skyline Technology Consulting Group, Znc., San Francisco, California 94133 (A.H.C.K.); and Genentech, Znc., South San Francisco, California 94080 (J.M.)

Norfloxacin, an orally active fluoroquinolone antimicrobial, has been reported to be embryolethal but not teratogenic when administered to pregnant cynomolgus macaques prior to gestational day (GD) 36 at doses ≤200 mg/kg/day. Additional studies have been performed in an effort to examine the mechanism responsible for this effect, particularly regarding the role of progesterone (P). The first study (Study I) investigated the effect of norfloxacin administration during early pregnancy (200 mg/kg/day; daily GD 20–30) in the absence of a functional corpus luteum (CL). The CL was surgically removed from 16 gravid females on GD 19 in order to focus on placental-derived P; ten were dosed with norfloxacin and six received vehicle only. Embryolethality was observed for 7/10 (70%) of the treated animals during GD 25–31 versus 0/6 (0%) for controls. A reduction in serum P was noted prior to embryonic loss, although no significant effects on chorionic gonadotropin (CG), 17β-estradiol (E2), or P or E urinary metabolites were observed. A second study (Study II) was performed in order to evaluate the capacity of norfloxacin (200 mg/kg) to reduce CL-derived P in both normally cycling and CG-stimulated nonpregnant females (ten treated, ten controls; daily for 8 days). No effects on P production or on luteal phase or menstrual cycle lengths were observed. The third study (Study III) was designed to examine the effect of norfloxacin on the metabolism and excretion of P in nonpregnant females. Silastic P implants were placed subcutaneously in order to maintain constant P levels during a 10 day treatment regimen (200 mg/kg/day; ten controls, nine treated). Five of the controls and four of the norfloxacin-treated females also received 14C-P intravenously within 1 hr of the last dose of norfloxacin in order to study excretory patterns. No significant differences between control and treated groups were observed. The results of these studies combined suggest that the developmental toxic effects observed in prior studies and Study I are specific to pregnancy and directly related to placental-derived P production. © 1992 Wiley-Liss, Inc.

The maternal pharmacokinetics, metabolism, and placental transfer of 13-cis-retinoic acid (isotretinoin) have been determined in the cynomolgus monkey using a dosing regimen which had been previously shown to result in retinoid-specific teratogenic effects [Hummler et al. (1990) Teratology 42: 263–272]. The drug (2.5 mg/kg body weight) was administered by nasogastric intubation once a day between gestational days (GD) 16–26, and twice a day between GD 27–31. Maternal plasma kinetics were determined following dosing on GD 26 and GD 31, and placental transfer was studied following the last dose on GD 31. The plasma half life of 13-cis-retinoic acid in the monkey (13.2 h) was comparable to that in the human. The main plasma metabolite in the monkey was the 13-cis-4-oxo-retinoic acid which occurred at levels lower or comparable to those of the administered drug. During multiple dosing, this metabolite accumulated to the same degree as the parent drug. All-trans-retinoic acid was present in maternal plasma in very low concentrations (2% of 13-cis-retinoic acid). The β-glucuronides of all-trans-and 13-cis-retinoic acid were further minor plasma metabolites. 13-cis-retinoic acid and its 4-oxo-metabolite reached the monkey embryo slowly but extensively during organogenesis and reached 24 h-AUC values of 956 and 590 ng·h/g embryo wet weight, resulting in embryo/maternal plasma concentration ratios of 0. 41 and 0. 33, respectively. The AUC value of alltrans-retinoic acid (316 ng·h/g) was only raised approximately 40% above the endogeneous AUC level (225 ng·h/g); only at two time periods examined were the embryonic all-trans-retinoic acid concentrations above endogeneous levels (at 4 h and 8 h; P < 0.01 and < 0.05, respectively; Student's t-test). The β-glucuronides of all-trans-and 13-cis-retinoic acid were not detected in the embryo. Accumulation of 13-cis-retinoic acid and the 4-oxo-metabolite during the twice-per-day dosing regimen was apparent both in maternal plasma and embryo. An interspecies comparison suggests that the half life as well as the metabolic pattern of 13-cis-retinoic acid in plasma were similar in monkey and human: 13-cis-4-oxo-retinoic acid was the main metabolite in both species and the β-glucuronides as well as the all-trans-retinoic acid were minor metabolites. However, the plasma AUC values of the administered drug and particularly the 4-oxo-metabolite were found to be lower in the monkey as compared to the human. This is one important fact why higher doses are needed in the monkey than in the human to elicit a teratogenic response. Our results indicate that the cynomolgus monkey is a good model for the human in regard to 13-cis-retinoic acid teratogenesis: we provide strong evidence that the high sensitivity of the monkey in comparison to other animal species (in rats and mice 20–25-fold higher doses are needed to elicit a teratogenic response) is due to prolonged and pronounced maternal and embryonic exposure to the parent compound and its 4-oxo-metabolite in the monkey embryo, which are implicated as proximate teratogenic agents. © 1994 Wiley-Liss, Inc.

The multiple applications of diagnostic ultrasound in obstet- rics have resulted in a continued rise in the prenatal population exposed each year. Although human epidemiologic and experimental studies with various animal models have not consistently documented any significant, reproducible findings related to clinically relevant exposures, technologic changes in scan- ning equipment and gaps in our knowledge regarding the interactiods) of ul- trasound with tissues emphasize the need to pursue safety issues. Studies with nonhuman primates have provided information on the potential for pre and postnatal effects on offspring exposed repeatedly during gestation (ATL MK 600, 7.5 MHz, Ism* = 27 mW/cmz; IsrpA = 85 Wicm'; Estimated power = 12 mW-scanned for 10 min 5 times weekly gestational day (GDl 20-35; 3 times weekly GD 36-60; once weekly for 20 min GD 60-1501. These studies have indicated transient effects on body weight, white blood cell counts (WBCs), and muscle tone postnatally. In an effort to confirm these findings and focus on hematologic changes, a second series of studies was initiated using the same exposure conditions (N = 22; 11 exposed, 11 sham controls). Data derived from both studies were combined and confirmed transient reductions in body weights for infants up through 4 months of age (P 5 0.03); no statistically significant differences in muscle tone were noted. Similar to the original findings, WBCs were transiently reduced on days 3 (P 5 0.02) and 21 (P 5 0.05); prenatal sampling indicated a significant difference between the groups on GD 140 (P 5 0.04). No direct effects were evident in bone marrow aspirates collected on postnatal days 3, 9, and 21 2 1. Although animals were able to compensate for these observed changes and remained unaffected by their occurrence, additional studies will be required to further our understanding of this phenomenon. 0 1993 Wiley-Liss, Inc.

The teratogenic potential of 13-cis retinoic acid (13-cis RA) was further assessed in cynomolgus macaques (Macaca fascicularis) following exposure to two different regimens during the preorganogenic period to determine the influence of time of treatment on the 13-cis RA malformation syndrome established previously (Hummler et al., Teratology, 42:263–272, 1990). In experiment (Exp) 1, 13-cis RA was orally administered once daily (2.5 mg/kg) on gestational day (GD) 16–25 and twice daily (2 × 2.5 mg/kg) on GD 26–27. In Exp 2, the same oral dose was administered once daily on GD 10–20 and twice daily on GD 21–24. Although the ear was affected at about the same incidence (6/9, 67%) in Exp 1 as previously reported, the defects were less severe. Except for hypoplastic vermis of the cerebellum (2/9, 22%), no other defects were seen. In Exp 2, the teratogenic effects of 13-cis RA on the craniofacial skeleton (1/9, 11%), external ear (5/9, 56%), and heart (2/9, 22%) were similar to that reported by Hummler et al.; however, no thymus or cerebellar malformations were observed. Analysis of the three different treatments (GD 16–27 in Exp 1, GD 10–24 in Exp 2, and GD 10–27 in Hummler et al.) suggests that the sensitive periods in the macaque are 1) craniofacial skeleton and heart GD 10–24, 2) thymus and cerebellum >GD 24, and 3) external ear GD 16–24, although the defects are less severe following the shorter exposure. Exposure to 13-cis RA preceding onset of morphological organogenesis appears to be a requirement for 13-cis RA embryopathy in the cynomolgus monkey. The external ear appears to be both a uniquely sensitive biomarker for 13-cis RA teratogenicity and an indicator of embryotoxic severity. Finally, the longer period of drug administration (2.5 mg/kg, GD 10–25, and 2 × 2.5 mg/kg, GD 26–27) used by Hummler et al. is more suited to assessment of 13-cis RA teratogenicity in macaques due to greater concordance with the 13-cis RA syndrome in humans. © 1993 Wiley-Liss, Inc.

The embryotoxic and teratogenic potential of all-trans retinoic acid was assessed following exposure prior to and during early organogenesis in the cynomolgus monkey (Macaca fascicularis). Sixteen pregnant females were orally administered all-trans retinoic acid (Tretinoin, Hoffmann-La Roche) once daily from GD 10-20 and twice daily from GD 21-24 at three different dosages, 5 (n = 9), 10 (n = 6) and 20 mg/kg (n = 1). Adverse clinical signs resembling hypervitaminosis A were observed in one animal at 5 mg/kg, in three animals at 10 mg/kg, and in the animal treated with 20 mg/kg all-trans retinoic acid. Maternal weight loss was observed in the 10- and 20-mg/kg groups. A dose-dependent increase in embryolethality was observed, with 22% (2/9), 50% (3/6), and 100% (1/1) occurring at 5, 10, and 20 mg/kg, respectively. The majority of embryonic deaths occurred between GD 16 and 20; the incidence of these early losses was higher than in historical and concurrent controls. No malformations, but a single growth-retarded fetus, was observed in the 5-mg/kg group. Craniofacial malformations, consisting of external ear defects, mandibular hypoplasia, cleft palate, and temporal bone abnormalities, were seen in three viable fetuses in the 10-mg/kg group. Skeletal variations were common to the majority (70%, 7/10) of viable fetuses in both dose groups and were increased relative to historical controls (32%, 25/77). Unlike previous studies with 13-cis-retinoic acid during the pre- and early organogenic stages of development (Hummler et al., Teratology 42:263-272, 1990), no thymic hypo- or aplasia or heart anomalies were observed, which may be attributable to the slightly longer 13-cis retinoic acid treatment period, i.e., GD 10-27. However, external ear and temporal bone defects were common to both all-trans and 13-cis retinoic acid. The similarity observed in the malformation syndrome induced by both all-trans and 13-cis retinoic acid in the cynomolgus monkey and 13-cis retinoic acid embryopathy in humans supports this macaque species as a model for further developmental toxicity studies of vitamin A-related compounds.

The embryotoxic and teratogenic potential of 13-cis retinoic acid was assessed in the cynomolgus macaque (Macaca fascicularis). A total of 41 animals was orally administered 13-cis retinoic acid in four sequential experiments. In Exp. 1 three dose levels, 2, 10, and 25 mg/kg, were administered on gestational day (GD) 18-28; 5 mg/kg was administered as an equally divided dose twice daily in Exp. 2 and 3 on GD 21-24 and on GD 25-27, respectively; in Exp. 4 the drug was administered at 2.5 mg/kg once daily from GD 10 to 25 and twice daily (2 x 2.5 mg/kg) on GD 26 and 27. Maternal death and toxicity, manifested as reduction in maternal weight and food consumption, and diarrhea, was observed in Exp. 1 in all three dose groups. No significant maternal toxicity was observed in the treatment groups in Exp. 2, 3, and 4 or in the control group. The primary manifestation of developmental toxicity was embryolethality in Exp. 1 and 2. The incidence of embryonic deaths in Exp. 3 was comparable to the historical controls. No malformations in GD 100 fetuses were observed in Exp. 1, 2, and 3. In Exp. 4, five of seven fetuses (71%) had malformations of both external ears, four of seven fetuses (57%) exhibited hypo- or aplasia of the thymus, and two of seven (29%) had malformations (transposition of the great vessels, ventricular septal defect) of the heart. The teratogenic dose for the cynomolgus monkey in the present study was lower than that reported for all other experimental species. Although central nervous system and craniofacial defects were not observed, the incidence of ear and thymus defects was similar to that reported for the human. The cardiovascular defects resembled those reported clinically, but the incidence was lower in the cynomolgus monkey. The similarity in teratogenic sensitivity to humans supports the use of the monkey as a model for developmental toxicity studies of vitamin A-related compounds.

The extensive use of ultrasonography for the prenatal assessment of growth and development continues to present questions regarding biological effects. We are currently evaluating a nonhuman primate model (Macaca fascicularis) exposed to ultrasound from gestational day (GD) 21 to 152 +/- 2. Exposures were performed with a commercial real-time sector scanner (ATL, MK 600); animals were scanned five times weekly on GD 21-35 +/- 2, three times weekly on GD 36-60 +/- 2, and once weekly on GD 61-150 +/- 2. The length of exposure was approximately the same as human exposure (GD 21-60 +/- 2 = 10 min/exam and GD 61-150 +/- 2 = 20 min/exam) although the frequency of the examinations was considerably greater. Initial reports indicated differences between control and treated animals including lower birth weight, higher simian Apgar scores, and changes in select hematologic parameters. Follow-up evaluations of growth during the first year included measurements of body weight, hand and foot lengths, humerus and femur lengths, biparietal and occipitofrontal diameters, head circumference, arm circumference, chest circumference, skinfold thickness, and crown-rump length. Results indicated a significant reduction in body weight in treated animals during the first three months, with nonsignificant differences during the following nine months. Hematologic analysis including complete blood counts (CBC) and clinical biochemistry at 6, 9, and 12 months of age were not significantly different. A series of behavioral evaluations including a neurobehavioral test battery (NBT) and tests assessing motor and cognitive skills were included. The NBT revealed increased muscle tone in treated animals at one, two, and four days. In an observation cage (week 1-14) more quiet activities were displayed by treated animals. Group differences in performance of motor and cognitive tasks were observed and may be attributable to agitation and difficulties in adjusting to test environments. There were no group differences observed in discrimination learning. When considering the possible implications to the human population, it is important to consider the amount of exposure these animals received, and the fact that most of the effects observed appeared to be transitory. Although human epidemiological studies have not revealed any significant bioeffects, the "prudent use" of diagnostic ultrasound should still be kept in mind. This is especially significant with the current rise in the use of endovaginal scanning and pulsed Doppler.

Seventeen timed-mated bonnet monkeys (Macaca radiata) were exposed to hyperthermia in a forced-draft incubator for a duration of one, three, four or eight days between 21 and 46 days of gestation. On each day of exposure the core temperature of the pregnant animal was elevated 2.4 to 5.4°C above the reference temperature recorded before heat exposure. Malformations occurred following a one-hour daily elevation of the core temperature above the reference temperature under the following exposure conditions: 3.8°C, days 23 to 26; 3.9°C, days 24 to 27; 2.4°C, days 27, 28 and 30; and 4.1°C, day 26 of gestation. Five resorptions occurred following exposure for one, three or four days between days 24 and 29 of gestation after core temperature elevation of 3.7 to 4.4°C and three abortions occurred following exposure for four days between days 24 and 35 of gestation after core temperature elevation of 2.6 to 3.9°C. The results clearly indicate that the teratogenic sensitive period is from 23 to 30 days gestation following elevations of the core temperature from 2.4 to 4.1°C. No distinct pattern of malformations was observed although skeletal and umbilical cord malformations were the most common. Pathological changes in placental structure, expecially maternal surface infarctions and intervillous thrombi, were associated with three of the four abnormal fetuses, suggesting a relationship between pathological placental characteristics and fetal malformations in this study. The degree of core temperature elevation which resulted in malformations in the present study are similar to those applied to other mammalian species, and the specific anomalies observed in the bonnet monkey resemble those observed in other mammalian species, including man.

Gross malformations of the external, middle, and internal ear were seen in fetal monkeys following maternal ingestion of thalidomide. Twenty-five pregnant bonnet monkeys (Macaca radiata) were each given a single oral dose of 10 mg/kg of thalidomide on day 24, 25, 26, 27, 28, or 29, or 30 mg/kg on day 25 or 28 of gestation. (Day of mating is assumed to be day zero of pregnancy). The skeletons, processed and stained with Alizarin Red S, were examined for changes in configuration and/or ossification of the ossicles and temporal bones. Bilateral temporal bones of one case, treated on day 24, were sectioned at 10 microns and examined histologically. Twelve fetuses collected at 70 +/- 3 days of gestation showed no gross ear defects, while 10 out of 13 fetuses collected at 100 +/- 3 days of gestation had structural anomalies similar to those observed in humans. All malformations were severe in fetuses treated on day 24 of gestation and lessened in degree of severity with treatments on days 25-28. Fetuses treated on day 29 were normal. External ear anomalies included microtia with meatal atresia or stenosis, and varying degrees of auricular hypoplasia. Defects of the middle ear were predominantly hypoplasia of malleus, incus, and tympanic ring. Fused ossicles and cochlear and vestibular window abnormalities were only seen in animals treated on day 24. The internal ear exhibited petrosal hypoplasia and delayed ossification of the lateral aspect of the lateral and posterior semicircular canals. Cochlear, vestibular, semicircular, and transcapsular canal defects were confirmed histologically in one case.

This study further defines the craniofacial malformations induced by triamcinolone acetonide in the rhesus monkey. Ten timed-mated pregnant rhesus monkeys (Macaca mulatta) received intramuscular injections of 10 mg/kg TAC on days 23, 25, 27, 29, and 31 of gestation. Results of previous experiments with rhesus and bonnet monkeys and baboons indicated that specific craniofacial and brain malformations could be induced with TAC during this period of pregnancy (Hendrickx et al., '80). Stage-matched TAC-treated and control embryos (stages 17-18 and 22) and age-matched TAC-treated and control fetuses (50, 60, and 70 days gestation) were removed by hysterotomy. Stage 17-18 TAC embryos appeared grossly normal but histologic evaluation revealed a shortened anlage of the posterior cranial base. Stage 22 TAC embryos and all TAC fetuses exhibited craniofacial dysmorphia and encephalocele. The developing sphenoid was the earliest affected and most severely malformed bone. Its defects included reduced anterioposterior and transverse dimensions, reduced orbitosphenoid and alisphenoid, abnormal pituitary fossa, and reduced dorsum and tuberculum sellae. In addition, shortening of the posterior cranial base and decreased cranial base angle was a consistent finding in the treated embryos and fetuses. Decreased ossification and remodeling in the facial bones and abnormal position due to the malformed sphenoid occurred.

The objective of this investigation was to study the teratogenic effects of triamcinolone acetonide (TAC) in the rat. Pregnant females were injected intramuscularly once each day for three consecutive days (days 9–11, 12–14, or 15–17 of gestation) either with vehicle alone or with 0.125, 0.25, or 0.5 mg/kg TAC. All animals were euthanized on day 20 of gestation, and the fetuses were fixed for razor blade sectioning and skeletal examination. No maternal lethality was elicited in any of the treatment groups. There were decreases in fetal viability in the two higher dose groups treated on days 12–14 and the group treated with 0.5 mg/kg on days 15–17. These same three treatment groups showed significant increases in the percentage of malformed fetuses. The most prevalent malformation was cleft palate, with both complete and partial clefts being detected. Umbilical hernias and undescended testes were also found to be significantlymore frequent in the high-dose group treated on days 12–14. A reduced degree of ossification was found in all TAC-treated groups, but no specific skeletal malformations were detected. Fetal weight was significantly reduced in all TAC-treated groups. The results demonstrate that (1) TAC affects a variety of developmental processes resulting in resorption, cleft palate, umbilical hernias, undescended testes, reduced ossification, and fetal growth retardation, (2) these teratogenic effects are elicited at doses causing no maternal lethality, and (3) fetal growth retardation can be produced at doses below those required to produce malformations and during periods of gestation where no malformations are produced, supporting the use of this parameter as a sensitive indicator of embryotoxicity.

Eighteen pregnant Macaca mulatta, 15 Macaca radiata, and six Papio cynocephalus were treated with 5--20 mg/kg triamcinolone acetonide (TAC) between 21 and 43 days of gestation on single- or multiple-day treatment schedules. Prenatal deaths and stillbirths were tripled in the bonnet monkey and doubled in the rhesus monkey, but did not significantly increase in the baboon. The central nervous system and cranium were the most commonly malformed areas in all three species. The incidence of severe defects, e.g., cranium bifidum, encephalocele, meningocele, and hydrocephalus, was increased in multiple-day treated cases. Minor abnormalities such as aplasia cutis congenita, cranium bifidum occultum, and occipital lobe hypoplasia were more prevalent in single-day treated cases. The sensitive period (days 23--31) for TAC for this group of defects encompasses neural tube closure, rostral demarcation of the midbrain, and development of the primordial collicular plate and two midbrain neuromeres. The results of this study indicate that TAC is a valuable chemical tool for the study of malformations and pathogenesis of the brain and accompanying cranio-facial defects.

Norfloxacin, a new orally active antibiotic, was investigated in cynomolgus monkeys for potential developmental toxicity. Fifty-seven monkeys were administered a control vehicle or norfloxacin by nasogastric gavage during the major period of organogenesis on gestational days (GD) 21 through 50 at doses of 0, 50, 100, 150, or 200/300 mg/kg/day. There was no evidence of teratogenicity at any dose level. Maternotoxicity and a significant increase in embryolethality occurred following doses of 200/300 mg/kg/day. The maternotoxicity was not expected based on range-finding studies in nonpregnant female monkeys, which showed no signs of toxicity in doses up to 500 mg/kg/day. Additional studies were conducted to determine if norfloxacin caused similar toxicity later in gestation. Forty-six pregnant monkeys were dosed with a control vehicle or 200 mg/kg/day norfloxacin for one of three 10-day periods on GD 36-45, 71-80, or 111-120. There were no maternotoxic, embryotoxic, or fetotoxic effects observed. Plasma concentrations of norfloxacin in five cynomolgus monkeys following 50 and 200 mg/kg oral doses were not dose-proportionate. However, at a given dose, administered in cross-over fashion, plasma concentrations of norfloxacin were higher in nonpregnant females (approximately 20-40%) than during pregnancy when the same subject was compared. At the no-observed-effect dose for maternal and embryotoxicity (50 mg/kg), peak plasma concentrations of norfloxacin in pregnant cynomolgus monkeys are approximately threefold higher than those observed in human volunteers receiving norfloxacin at the maximum recommended therapeutic dose of 400 mg (5.7 mg/kg based on 70 kg body weight) twice per day.

Triamcinolone acetonide (TAC) was administered to pregnant macaques (Macaca mulatta [15] and M. radiata [7]) during gestational days (GD) 23 to 41 using various dosing schedules. A daily dose of 10 mg/kg is approximately equal to 100 x the human dose equivalent. The brains of the fetuses and infants were studied grossly and histologically. All cases displayed either the mild form of the TAC-induced syndrome (craniofacial dysmorphia, cranium bifidum occultum, meningocele, and mild distortion of the midbrain) or the more severe form (occipital encephalocele, hydrocephalus, severe distortion of the midbrain or midbrain "beaking," shunting of cerebrospinal fluid, and craniofacial malformations). The dysmorphology was dose-related, with severity increasing at higher doses or with increased numbers of treatments. Individual cases were assessed for the severity of the syndrome by comparison of like components between groups. The lesions observed were morphologically comparable to those described in spontaneous human cases; the TAC-induced occipital encephaloceles were associated with brainstem and cerebellar abnormalities, and, with the less severe form of the syndrome, brainstem abnormalities were occasionally present, with occipital meningoceles. Controversy exists concerning the significance and temporal development of the midbrain changes. However, the associated alteration in aqueduct conformation may have been responsible for functional compromise and ensuing hydrocephalus.

Two sex steroid compounds which have been used clinically for parenteral supportive therapy of pregnancy were examined for embryotoxic effects in rhesus and cynomolgus macaques. Hydroxyprogesterone caproate (HPC) alone or in combination with estradiol valerate (EV) were administered intramuscularly (i.m.) to pregnant monkeys at 7-day intervals between 20 and 146 days of gestation and fetuses were examined following cesarean section at 150 +/- 2 days. HPC alone was tested in both species at doses ranging from 0.01 X to 10 X the human dose equivalent (HDE); only rhesus monkeys were exposed to the HPC + EV combination at 0.1 X to 10 X HDE. Total embryolethality resulted following the administration of HPC alone and combined with EV at 1 X and 10 X HDE in rhesus monkeys; the level of abortions in cynomolgus monkeys exposed to HPC (0.1 X to 1 X HDE) was comparable to controls. A small number of nonspecific malformations and developmental variations observed in cynomolgus fetuses after HPC exposure were considered to be incidental findings. No anomalies were found in surviving rhesus monkey fetuses treated with HPC + EV. The results indicate that long-term in utero exposure to the progestin, HPC, alone or in combination with EV in rhesus and cynomolgus monkeys, is embryolethal but not teratogenic at doses up to ten times the human therapeutic dose.

Teratologic evaluation of a mild tranquilizer being developed for human therapeutic use involved daily oral administration to 29 baboons (P. cynocephalus) during organogenesis according to three treatment regimens. In Phase I, 9 animals (3 groups of 3) received 2, 6, or 20 mg/kg CG 3033 per day for 28 consecutive days between 18 and 45 days gestation; in Phase II, 14 animals (7 groups of 2) were given 20 mg/kg CG 3033 for four consecutive days: 18-21, 22-25, 26-29, 30-33, 34-37, 38-41, or 42-45; and in Phase III, 6 animals (2 groups of 3) were administered 40 mg/kg daily between days 18-21 or 22-25 of gestation. No teratologic changes attributable to drug treatment were observed, and the abortion rate was within the range for controls.

The frequency of use of ultrasonography for evaluating the developing embryo/fetus has continued to rise although the possible risks from exposure still remain uncertain. The cynomolgus macaque (Macaca fascicularis) is currently being used in our laboratory as a model to assess these risks. In utero exposure was performed utilizing a commercial real-time mechanical sector scanner with a 7.5 MHz scanhead (ATL, MK 600). Maximum acoustic power output for this unit is as follows: I(SPTA) = 12.0 mW/cm2, I(SPPA) = 98 W/cm2, and Im = 137 W/cm2. Animals exposed to ultrasound (N = 16) were scanned five times weekly on gestational days (GD) 21-35 +/- 2 for 10 minutes/exam (m/e), three times weekly on GD 36-60 +/- 2 for 10 m/e, and once weekly on GD 61-150 +/- 2 for 20 m/e. Controls (N = 14) were "scanned" with the unit placed on standby. Assessment of simian Apgar scores at 1, 5, and 10 minutes of life revealed higher scores for treated animals at 10 minutes (P less than or equal to 0.045); greater scores in muscle tone (P less than or equal to 0.013) and color (P less than or equal to 0.016) were observed. Evaluation of morphometrics at birth including weight, biparietal diameter, occipitofrontal diameter, head circumference, hand and foot lengths, humerus and femur lengths, arm circumference, chest circumference, tail length, skinfold thickness, and crown-rump length (CRL) indicated a significant reduction in only two parameters, birth weight (P less than or equal to 0.027) and CRL (P less than or equal to 0.033). Hematologic analysis at 2 +/- 1, 9 +/- 1, and 16 +/- 1 days of life revealed a significant difference in white blood cell counts (WBCs). Treated animals displayed lower WBCs with reductions in numbers of segmented neutrophils and monocytes at all ages observed. Hematologic differences were not significant by 5-6 months of age. No abortions, gross malformations, or stillbirths were observed in the exposed animals.

A single dose of MPA (Depo-Provera; Upjohn Co., Kalamazoo, Michigan) was administered intramuscularly to 12 time-mated pregnant cynomolgus monkeys on day 27 (+/- 2) of gestation at 25 mg/kg or at 100 mg/kg. Maternal blood samples were collected immediately prior to MPA injection and then at regular intervals until cesarean section at term (day 152 +/- 3). Infants in both dose groups had external genital abnormalities. Female infants in the low-dose groups had partial or complete labial fusion, prominent median raphe, and clitoral hypertrophy; at high doses (100 mg/kg), the female infants had complete labial fusion and a distinct penile urethra. MPA had an opposite effect on external genitalia of male infants. The penis was short and the scrotal swelling was absent or less conspicuous, and two males had hypospadias. The adrenal glands were significantly smaller (P less than 0.05) in infants of both sexes treated with 100 mg/kg. One of the infants treated with 25 mg/kg of MPA had a muscular ventricular septal defect. Serum concentrations of MPA were determined by radioimmunoassay in eight pregnant monkeys. In the 25 mg/kg group the patterns of MPA profiles in the serum were similar in all four animals. An initial peak occurred at 24-48 hr postinjection (2.7-9.6 ng/ml), followed by a slight decrease at 3 days postinjection (gestational day 30), and then a steady increase to maximum levels of 10-14 ng/ml occurring between gestational days 37 and 50. Serum levels gradually declined to concentrations below 5 ng/ml by midgestation in three of four monkeys. By comparison, both the patterns and magnitude of MPA concentration showed great interanimal variation in the 100 mg/kg group. MPA was present in cord blood at measurable concentrations in infants at both dose groups; the levels ranged from 0.6 to 8.3 ng/ml, corresponding to 40-72% of the maternal concentrations. These results demonstrate that a single injection of MPA during early pregnancy causes selective embryotoxicity in both male and female fetuses. Presence of high levels of MPA in maternal sera during the critical period of genital development can cause specific genital defects; however, the exact mechanism by which MPA causes these paradoxical genital abnormalities is unknown.

Fifteen pregnant Macaca mulatta were treated with doses of 20 or 40 mg of retinoic acid between 19--45 or 17--45 days of gestation, respectively, for 4--8 consecutive days. Based on gross examination, ten malformed infants, including one stillbirth and one abortus, four normal infants, and one resorption were produced. The most critical sensitive period was between days 24--35 of gestation, and the malformations primarily involved the craniofacial skeleton. Ten treated infants and eight age-matched controls were cephalometrically analyzed using craniometric points as closely correlated as possible with those in humans in order to define the craniofacial malformations induced prenatally by retinoic acid. Although all ten animals had detectable linear and angular deviations from the controls, four had cephalometric patterns which appeared to be of similar developmental origin.

Cynomolgus monkeys, rhesus monkeys and baboons were administered 10 to 40 times the human dose equivalent of Bendectin throughout the major period of organogenesis (22(+/-3)-50 days of gestation). In animals examined prenatally (100 +/- 2 days gestation) the total incidence of ventricular septal defects (VSD) was 40% in cynomolgus monkeys, 18% in rhesus monkeys, and 23% in baboons. The majority of VSD involved the muscular portion of the septum. No dose response was evident and there were no other cardiac or extracardiac defects found except for one baboon fetus with multiple defects. No defects were observed in cynomolgus monkeys administered Bendectin for 4-day periods between 22 and 41 days of gestation. There was no association of Bendectin treatment with any noncardiac defect. In cynomolgus and rhesus monkeys examined at term there was one mitral valve defect and no incidence of VSD. The increased incidence of VSD observed prenatally in all three species and the absence of defects in macaques at term suggests a delay in closure of the ventricular septum in treated animals. The Bendectin-treated monkey may be a suitable model for the study of the pathogenesis of VSD and the mechanism of spontaneous closure of the defect.

Two sex steroid hormone combinations which have been used clinically as tests for detection of early pregnancy were examined for embryotoxic effects in macaques and baboons. Norethisterone acetate and ethinyl estradiol (NEA + EE) were orally administered to rhesus and cynomolgus monkeys and baboons at dosages ranging from one to 1,000 times the human dose equivalent (HDE) during days 20–50 of pregnancy. Progesterone and estradiol benzoate (P + EB) were delivered by two to six intramuscular injections to rhesus and cynomolgus monkeys between gestational days 20 and 35 at 0.1–25 × HDE. Fetuses were examined following cesarean section at 100 ± 2 days (NEA + EE) or at term (P + EB). The results showed increased embryolethality over controls at 100–1,000 × HDE (NEA + EE) and at 10 and 25 × HDE (P + EB). Besides growth retardation, isolated cases of minor nongenital malformations were observed only in cynomolgus monkeys following treatment with both hormone combinations mainly at embryolethal dose levels and were considered spontaneous in nature. Virilization of female cynomolgus fetuses following NEA + EE treatment was manifested as two cases of clitoral enlargement in the 300 × HDE group and two cases of increased anogenital distance with reduced vaginal opening in the 1,000 × HDE group. The highest dose of NEA + EE was also maternally toxic, as two maternal deaths occurred at the end of the treatment period. One dead female cynomolgus fetus exposed to P + EB (10 × HDE) also exhibited masculinized external genitalia. The results support epidemiologic and laboratory data which indicate that sex steroid exposure during early pregnancy does not induce nongenital teratogenicity.

Triamcinolone acetonide (TAC) is teratogenic in rats while cortisol has been reported as not teratogenic. The objective of this investigation was to determine whether this difference in teratogenicity could be due to a difference in the metabolism and distribution of the parent compound in the embryomaternal unit. 3H-TAC and 14C-cortisol were administered intramuscularly to pregnant rats on day 12 of gestation. These dams were killed at each of the following time points after injection: 0.5, 1, 3, 6 and 24 hr. Maternal plasma and embryos were analyzed by high performance liquid chromatography (HPLC) and liquid scintillation counting. The plasma concentration of parent TAC was significantly greater than that for parent cortisol at all time points. The plasma elimination half-life for TAC, 86 min, was also calculated to be significantly longer than that for cortisol, 8 min. Furthermore, the percentage of total plasma radioactivity representing HPLC resolved TAC was much higher than that representing cortisol at all time points. The concentration of TAC in the embryos was significantly greater than for cortisol at all time points. The elimination half-life for unchanged TAC in the embryos was 142 min compared to 22 min for cortisol. The percentage of total radioactivity in the embryos representing unchanged TAC was similar to that found in maternal plasma while the percentage of total radioactivity representing unchanged cortisol was much lower than that found in maternal plasma. These findings support the hypothesis that differences in the distribution and metabolism of the parent compound are a critical factor in determining the teratogenicity of that compound.

Imipramine hydrochloride was administered orally twice daily to 18 bonnet and 3 rhesus monkeys between days 23 and 45 of pregnancy for 1-3 or 18-22 days at 1, 2, and 10 times the recommended human dose. No teratologic changes were observed, although signs of maternal toxicity occurred at the high dose level, and the abortion rate was higher than in controls.The possible teratologic effect of imipramine hydrochloride (IHC) was investigated in pregnanct bonnet and rhesus monkeys. Oral IHC was administered twice daily between Days 23-45 of pregnancy, for either 1-3 days or 18-22 days, at 1, 2, and 10 times the recommended human dose. No teratogenic effects were observed with either acute or chronic treatment. The incidence of early abortion was 9% in the treated groups, compared with 3% in controls. Clinical signs of maternal toxici ty were observed at the high dose level, though symptoms disappeared within 24 hours of the last treatment. It is concluded that the teratogenic potential of IHC is low in humans when used at recommended doses.

This study was undertaken to assess the developmental toxicity and drug distributional and metabolic characteristics of prenatal valproic acid (VPA) exposure in rhesus monkeys. Oral administration of 20-600 mg/kg/day VPA (approximately 1-15 X human therapeutic dose) to 33 animals on variable gestational days (GD) during organogenesis resulted in dose-dependent developmental toxicity manifested as increased embryo/fetal mortality, intrauterine growth retardation, and craniofacial and skeletal defects. Biphasic plasma elimination curves were observed for total and free VPA on the first (GD 21) and last (GD 50) days of treatment in the 100- and 200-mg/kg/day dose groups. VPA exhibited dose-independent elimination kinetics at the plasma concentrations observed in this study. There was no significant change in pharmacokinetic parameters (maternal plasma elimination rate, area under the curve, peak plasma concentration) between the first and last days of treatment at either dose level. Placental transfer studies indicated that embryos were exposed to half the free VPA concentrations present in maternal plasma on GD 37. Comparisons of interspecies sensitivity to VPA-induced developmental toxicity in the mouse, rat, monkey, and man are made.

The embryotoxic and teratogenic potential of Bendectin was assessed in this double-blind study in the cynomolgus monkey (Macaca fascicularis). Bendectin was administered orally at doses approximately two, five, and 20 times the human dose equivalent from 22 +/- 2 through 50 days of gestation. Fetuses were delivered by cesarean section near term and examined for malformations. There was no maternal toxicity as evidenced by maternal weights and physical signs. There was no correlation between dosage and the number of prenatal deaths. No significant abnormalities related to treatment were observed in postdelivery physical examinations, placental evaluations, external and internal gross examinations, or from radiographs of the neonates. Under the conditions of this study the treatment of pregnant cynomolgus monkeys with Bendectin produced no evidence of teratogenicity or embryo-, or fetal-, or maternal toxicity.

A controlled breeding program was used to collect baboon embryos of known insemination ages. Serial sections of the palatine region of 37 embryos and fetuses from 30 to 64 days (d) (estimated fertilization age) werestudied microscopically and by graphic or wax reconstruction. One 100-day fetuswas partially dissected. Nasal placodes appeared in Stage XIV (30 d, 4.3–6.0 mm).By Stage XVI (33–36 d, 6.9–9.0 mm) nasal pits developed. The primary palate and palatine processes were present at Stage XVII (36 d, 11.3–11.6 mm). Subsequent development produced rostrodorsal and vertical lengthening of the palatine processes. Rostrally they slanted caudomedially toward the base of the tongue, and dorsally they remained vertically oriented lateral to the tongue in Stage XXI (43–45 d, 18.3–19.6 mm). In Stage XXII (47 d, 22–24 mm) the midpalatine processesbegan to rotate to a horizontal position. By Stage XXIII (45–50 d, 25.8–28.0 mm)the rostral two-thirds of the palatine processes were horizontally oriented cranialto the tongue. They were fusing to each other and to the nasal septum. The dorsalone-third of the palatine processes were assuming a horizontal position by medialprojection. In the 53-d fetuses (39.6–48.0 mm) the hard and soft palates wereformed except for the uvula, which remained divided until sometime after 64 d(63.0 mm). Epithelial remnants were not formed in the soft palate, suggestingthat this area was formed by mesenchymal merging without epithelial fusion.

Twenty-nine pregnancies were obtained in 21 female bonnet monkeys. The females were housed individually and were mated for a 2-h period, usually 2 days before midcycle. The day of mating was designated as day o of pregnancy. After pregnancy was confirmed by rectal palpation the animals each received 5, 10, or 30 mg/kg thalidomide orally, either as single doses on days 24, 25, 26, 27, 28, or 29 or once per day on days 25–27 or 41–44. With 5 and 10 mg/kg only malformations of the forelimbs were observed, characterized by total absence (amelia) or partial absence (meromelia) of the free limb. In all cases the malformations were more severe on the left side than the right. No craniocaudal developmental gradient was noted with 10 mg/kg. With 30 mg/kg forelimb and hindlimb malformations occurred, although the hindlimb malformations were mild and consisted essentially of either bowing or partial absence of the femur. A craniocaudal developmental gradient was evident with 30 mg/kg. Treatment during the time of palate closure (days 41–44) did not affect normal palate development. Absence and/or malformations of the uterus, ovaries, kidney, heart, brain, spleen, and umbilical arteries also occurred.