Exposure of cynomolgus monkey embryos to retinoic acid causes thymic defects: effects on peripheral lymphoid organ development.

We have previously reported that exposure of monkey embryos to 13-cis-retinoic acid (cRA) results in thymic defects. In this study, we analyzed lymphocyte and antigen-presenting cell populations at gestational days (GDs) 80-100 in the thymus, spleen, mesenteric lymph nodes, and gut-associated lymphoid tissue following a teratogenic dosing regimen of cRA (2.5 and 5 mg/kg) at GD14-27. Tissue sections were immunostained for T-cells (anti-CD3), B-cells (anti-CD20), dendritic cells (p55), and major histocompatibility class II (anti-HLA-DR). Digital images of spleen sections were analyzed to obtain the relative area occupied by the cell subsets within the white pulp (WP). Compared with controls, the T-cell dependent compartment of the spleen WP in specimens with perturbed thymic development (aplasia and severe hypoplasia) showed a reduction in size and proportion of CD3(+) T cells. Our findings indicate that cRA-induced thymic defects result in disrupted development of the splenic T-cell dependent compartment.

Two cases of digital defects in Macaca mulatta infants and a survey of the literature.

Although congenital digital defects, particularly polydactyly, have been reported frequently in humans, their occurrence in rhesus macaques is relatively rare. We observed two cases of spontaneous digital defects in male rhesus monkey infants recently born at the California Regional Primate Research Center. One infant exhibited bilateral postaxial polydactyly and the other infant had bilateral oligodactyly with unilateral phalangeal duplication. In this report, we present the clinical/pathological details of these cases as well as discuss the embryology of normal and abnormal limb development. We will also summarize a variety of spontaneous and experimentally induced digital defects that have been reported in several nonhuman primate species.

Structures of bovine glutamate dehydrogenase complexes elucidate the mechanism of purine regulation.

Glutamate dehydrogenase is found in all organisms and catalyses the oxidative deamination of l-glutamate to 2-oxoglutarate. However, only animal GDH utilizes both NAD(H) or NADP(H) with comparable efficacy and exhibits a complex pattern of allosteric inhibition by a wide variety of small molecules. The major allosteric inhibitors are GTP and NADH and the two main allosteric activators are ADP and NAD(+). The structures presented here have refined and modified the previous structural model of allosteric regulation inferred from the original boGDH.NADH.GLU.GTP complex. The boGDH.NAD(+).alpha-KG complex structure clearly demonstrates that the second coenzyme-binding site lies directly under the "pivot helix" of the NAD(+) binding domain. In this complex, phosphates are observed to occupy the inhibitory GTP site and may be responsible for the previously observed structural stabilization by polyanions. The boGDH.NADPH.GLU.GTP complex shows the location of the additional phosphate on the active site coenzyme molecule and the GTP molecule bound to the GTP inhibitory site. As expected, since NADPH does not bind well to the second coenzyme site, no evidence of a bound molecule is observed at the second coenzyme site under the pivot helix. Therefore, these results suggest that the inhibitory GTP site is as previously identified. However, ADP, NAD(+), and NADH all bind under the pivot helix, but a second GTP molecule does not. Kinetic analysis of a hyperinsulinism/hyperammonemia mutant strongly suggests that ATP can inhibit the reaction by binding to the GTP site. Finally, the fact that NADH, NAD(+), and ADP all bind to the same site requires a re-analysis of the previous models for NADH inhibition.

13-cis-retinoic acid causes patterning defects in the early embryonic rostral hindbrain and abnormal development of the cerebellum in the macaque.

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.

Conjoined twins in a rhesus monkey (Macaca mulatta).

Conjoined twinning is an extremely rare occurrence among human and nonhuman primates. The current report describes a case of minimally conjoined omphalopagus rhesus monkey twins that were observed in the breeding colony at the California Regional Primate Research Center. The full-term nonviable male twins were morphologically normal and united in the umbilical region, involving the liver, xiphoid, umbilicus, body wall, and skin. The umbilical cords were separate, but closely aligned, within an ensheathing amnion; each cord contained two arteries and a vein. The closely associated cords were centrally inserted on a large bidiscoid placenta. There were no gross or histologic abnormalities in the viscera, with the exception of enlargement of the left central liver lobes, which constituted the area of attachment. The cause of death was attributed to asphyxia and trauma experienced during parturition.

Pathogenesis of retinoic acid-induced ear malformations in primate model.

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.

The structure of bovine glutamate dehydrogenase provides insights into the mechanism of allostery.

BACKGROUND: Bovine glutamate dehydrogenase (boGDH) is a homohexameric, mitochondrial enzyme that reversibly catalyzes the oxidative deamination of L-glutamate to 2-oxoglutarate using either NADP(H) or NAD(H) with comparable efficacy. GDH represents a key enzymatic link between catabolic and biosynthetic pathways, and is therefore ubiquitous in both higher and lower organisms. Only mammalian GDH exhibits strong negative cooperativity with respect to the coenzyme, however, and is regulated by a large number of allosteric effectors. RESULTS: The atomic structure of boGDH in complex with NADH, glutamate, and the allosteric inhibitor GTP has been determined to 2.8 A resolution. The major difference between the bacterial and bovine GDH structures is the presence of an additional 'antenna' in boGDH that protrudes from each trimer, twisting counterclockwise along the threefold axis. NADH and glutamate are clearly observed in the active site, but the contacts differ slightly from those observed in Clostridium symbiosum GDH. A second, inhibitory NADH molecule lies buried in the core of the hexamer. Finally, two GTP molecules bind near the hinge region connecting the NAD(+)- and glutamate-binding domains. CONCLUSIONS: We propose that the antenna serves as an intersubunit communication conduit during negative cooperativity and allosteric regulation. GTP and NADH inhibit GDH by keeping the catalytic cleft in a closed conformation. In contrast, ADP probably binds to the back of the NAD(+)-binding domain and activates the enzyme by keeping the catalytic cleft open. Extensive contacts between antennae within the crystal lattice may represent hexamer interactions in solution and, perhaps, with other enzymes within the mitochondrial matrix.

13-cis-Retinoic acid alters neural crest cells expressing Krox-20 and Pax-2 in macaque embryos.

This study investigates hindbrain and associated neural crest (NCC), otocyst, and pharyngeal arch development in monkey embryos following teratogenic exposure to 13-cis-retinoic acid (cRA). cRA was orally administered (5 mg/kg) to pregnant long-tailed macaques (Macaca fascicularis) between gestational days (GD) 12 and 27. Embryos were surgically collected at desired stages during treatment, analyzed for external morphological changes, and processed for immunohistochemistry. Two transiently expressed nuclear proteins, Krox-20 and Pax-2, were used as markers for the target cellular and anatomical structures. Rhombomere (r) expression patterns of Pax-2 (r4/r6) and Krox-20 (r3/r5) were maintained after cRA treatment, but r4 and r5 were substantially reduced in size. In untreated embryos, Krox-20 immunoreactive NCC derived from r5 migrated caudally around the developing otocyst to contribute to the third pharyngeal arch mesenchyme. In cRA-treated embryos, a subpopulation of NCC rostral to the otocyst also showed Krox-20 immunoreactivity, but there was a substantial reduction in Krox-20 post-otic NCC. Pax-2 immunoreactive NCC migrating from r4 to the second pharyngeal arch were substantially reduced in numbers in treated embryos. Alteration in the otic anlage included delayed invagination, abnormal relationship with the adjacent hindbrain epithelium, and altered expression boundaries for Pax-2. cRA-associated changes in the pharyngeal arch region due to cRA included truncation of the distal portion of the first arch and reduction in the size of the second arch. These alterations in hindbrain, neural crest, otic anlage, and pharyngeal arch morphogenesis could contribute to some of the craniofacial malformations in the macaque fetus associated with exposure to cRA.

Effects of 13-cis-retinoic acid on hindbrain and craniofacial morphogenesis in long-tailed macaques (Macaca fascicularis).

Hindbrain and craniofacial development during early organogenesis was studied in normal and retinoic acid-exposed Macaca fascicularis embryos. 13-cis-retinoic acid impaired hindbrain segmentation as evidenced by compression of rhombomeres 1 to 5. Immunolocalization with the Hoxb-1 gene product along with quantitative measurements demonstrated that rhombomere 4 was particularly vulnerable to size reduction. Accompanying malformations of cranial neural crest cell migration patterns involved reduction and/or delay in pre- and post-otic placode crest cell populations that contribute to the pharyngeal arches and provide the developmental framework for the craniofacial region. The first and second pharyngeal arches were partially fused and the second arch was markedly reduced in size. The otocyst was delayed in development and shifted rostrolaterally relative to the hindbrain. These combined changes in the hindbrain, neural crest, and pharyngeal arches contribute to the craniofacial malformations observed in the retinoic acid malformation syndrome manifested in the macaque fetus.

Crystallization and characterization of bovine liver glutamate dehydrogenase.

Bovine liver glutamate dehydrogenase has been crystallized as an abortive complex with glutamic acid, NADH, and an inhibitor, GTP. Crystals of this complex were grown using the sitting drop vapor diffusion method with PEG 8000 as the precipitant and diffract to better than 2.5 A resolution. The crystals belong to the space group P2(1) with an entire enzyme hexamer in the crystallographic asymmetric unit. Self-rotation and self-Patterson functions clearly define the orientation and position of this hexameric enzyme.

Perspectives on the use of the baboon in embryology and teratology research.

This paper summarizes the developmental stages of the baboon during the period of organ formation and provides comparative information for other primates, including the human. Special attention is directed to the early development of the nervous system, eye, ear and nose/palate. The similarity in development of these structures with humans indicates that the baboon is a suitable model for studies of normal and abnormal neurological development. Spontaneous prenatal loss rates in the baboon (2.4-11.2%) are slightly lower than those reported in rhesus and cynomologus monkeys. The baboon, in addition to the cynomologus monkey and macaque, has been used as a model in teratology research to assess the potential risk of thalidomide, sex steroids, Bendectin and rubella virus, as well as to study the pathogenesis of malformations associated with the corticosteroid triamcinolone acetonide. The rate of spontaneous malformations (<1%) in baboons, similar to that reported for other commonly used primates, supports their continued use as a teratological model. In this regard, a sample protocol is provided for the safety evaluation of biotechnology products using nonhuman primates, which are the most appropriate model for those compounds which are bioactive in species closely related to humans.

From blastocyst to placenta: the morphology of implantation in the baboon.

Implantation and placentation in the baboon share many morphological features with other primates, as well as having some specific distinctions. The ability to use deturgescence of the sex skin as a method of timing ovulation and the ease with which the uterine lumen can be flushed have been used to examine morphological aspects of blastocyst differentiation and implantation in this species. Preimplantation blastocysts were obtained by non-surgical flushing of the uterus 6-8 days after ovulation, and implantation sites were excised from uteri removed on days 10-16 of gestation. All tissues were prepared for electron microscopy by aldehyde fixation and plastic embedding. Maturation of trophoblast from the compacted morula stage to the expanded blastocyst stage includes increase in numbers of polyribosomes, changes in conformation of mitochondria, and development of an effective endocytic apparatus. An endodermal layer forms beneath the inner cell mass prior to loss of the zona pellucida, and parietal endodermal cells extend beyond the inner cell mass. Azonal blastocysts have regions of syncytial trophoblast adjacent to the inner cell mass, and they may represent adhesion stages of early implantation. In early postimplantation stages, trophoblast replaces the uterine epithelium and processes of syncytial trophoblast invade dilated superficial maternal vessels. In subsequent lacunar stages there is rapid elevation of the developing conceptus above the uterine surface as the lacunae enlarge. Cytotrophoblast rapidly enters maternal vessels, and arterioles are partially or completely occluded by migrating cytotrophoblast. The early access to controlled maternal blood flow apparently allows trophoblastic lacunae to expand superficially as opposed to more extensive endometrial invasion.

Frequency of spontaneous congenital defects in rhesus and cynomolgus macaques.

This paper summarizes the spontaneous incidence of congenital defects in the rhesus and cynomolgus macaque colonies (Macaca mulatta and M. fascicularis) at the California Regional Primate Research Center. The computerized database used in this analysis included fetuses, term infants, juveniles, and adults that underwent a necropsy procedure over a 14-year period (1983-1996). The calculated malformation rates were 0.9% (40/4,390) and 0.3% (3/965) for the rhesus and cynomolgus monkey, respectively. Most of the observed malformations in both species affected the musculoskeletal and the cardiovascular systems, while a smaller number of defects were observed in the gastrointestinal, urogenital, endocrine, and central nervous systems. Inbreeding did not contribute to the spontaneous malformation incidence and there was no predilection for sex (male vs. female) or housing (indoors vs. outdoors) among the malformed cases. This spontaneous malformation database in our macaque colony aids in the interpretation of defects that occur in an experimental study as well as in the ongoing assessment of a healthy nonhuman primate breeding colony.

Retinoid metabolism and transplacental pharmacokinetics in the cynomolgus monkey following a nonteratogenic dosing regimen with all-trans-retinoic acid.

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.

Analysis of hindbrain neural crest migration in the long-tailed monkey (Macaca fascicularis).

Neural crest cells make a substantial contribution to normal craniofacial development. Despite advances made in identifying migrating neural crest cells in avian embryos and, more recently, rodent embryos, knowledge of crest cell migration in primates has been limited to what was obtained by conventional morphological techniques. In order to determine the degree to which the nonhuman primate fits the mammalian pattern, we studied the features of putative neural crest cell migration in the hindbrain of the long-tailed monkey (Macaca fascicularis) embryo. Cranial crest cells were identified on the basis of reported distributional and morphological criteria as well as by immunocytochemical detection of the neural cell adhesion molecule (N-CAM) that labels a subpopulation of these cells. The persistent labeling of a sufficient number of crest cells with antibodies to N-CAM following their exit from the rostral, pre-otic and post-otic regions of the hindbrain facilitated tracking them along subectodermal pathways to their respective destinations in the first, second and third pharyngeal arches. Peroxidase immunocytochemistry was also employed to localize laminin and collagen-IV in neuroepithelial basement membranes. At stage 10 (8-11 somites), crest emigration occurred in areas of unfused neural folds through focal disruptions in the neuroepithelial basement membrane in both the rostral and pre-otic regions, although there was little evidence of crest migration in the post-otic hindbrain. By stage 11 (16-17 somites), the neural folds were fused (pre- and post-otic hindbrain) or in the process of fusing (rostral hindbrain), yet crest cell emigration was apparent in all three areas through discontinuities in the basement membrane. Emigration was essentially complete at stage 12 (21 somites) as indicated by nearly continuous cranial neural tube basement membranes. At this stage the pre-ganglia (trigeminal, facioacoustic and glossopharyngeal) were consistently stained with N-CAM. The current study has provided new information on mammalian neural crest in a well-established experimental model for normal and abnormal human development, including its use as a model for the retinoic acid syndrome. In this regard, the current results provide the basis for probing the mechanisms of retinoid embryopathy which may involve perturbation of hindbrain neural crest development.

Emigration of neural crest cells from macaque optic vesicles is correlated with discontinuities in its basement membrane.

It is established that cranial neural crest cells play critical roles in normal development, but the production of neural crest cells from the prosencephalon has received little attention, especially in primates. We therefore investigated the emigration into adjacent mesenchyme of neuroepithelial cells from macaque optic vesicles. Paraffin sections prepared from 13 embryos (Macaca fascicularis) representing developmental stages 10-14 were examined following standard immunoperoxidase staining for laminin and type IV collagen. At stage 10 the optic vesicle basement membrane was closely applied to that of the surface ectoderm except at its posterior border, where it contacted mesenchyme. The basement membrane was continuous and showed no evidence of cell migration. By stage 11 the optic vesicle basement membrane exhibited numerous gaps along its posterior border. These defects were frequently occupied by cells emigrating from the optic vesicle epithelium, as judged by the deflection of basement membrane fragments. Gaps were also seen along the lateral border, where migrating neuroepithelial cells were positioned between the surface ectoderm and optic vesicle. This cell migration appeared to increase during stage 12, with deterioration of all areas of the optic vesicle basement membrane. Basement membranes of the surface ectoderm, adjacent mesencephalon, and telencephalon anlage remained intact. By stage 13 the basement membranes of the optic vesicles were repaired and nearly continuous, with migrating cells rarely seen. Development of the optic cups at stage 14 indicated a near absence of basement membrane defects and emigrating cells.

Intracellular pH monkey embryos at various stages of organogenesis estimated by dimethadione distribution.

Previous experiments using the transplacental distribution of 14C-DMO (5,5-dimethyloxazolidine-2,4-dione or commonly known as dimethadione) have demonstrated that the pH of rat embryos and fluids progressively decreases during organogenesis. The aim of the present experiments was to similarly evaluate pH changes during organogenesis in the cynomolgus monkey, which is a model for human embryogenesis. Using DMO quantitated by gas chromatography-mass spectrometry as opposed to the counting of radiolabelled compound, cynomolgus monkey embryos were determined to undergo a similar decrease in embryonic pHi over an approximately comparable period of development (Days 24-36 of gestation). The ratio of DMO in chorionic fluid to DMO in maternal plasma in the cynomolgus monkey also displayed a decrease with advancing gestational age indicative of a pH decrease. The DMO transplacental distribution was found to be significantly slower in the cynomolgus monkey than that in rodents. The present investigation indicates that the magnitude of the reduction of pH in embryonic cells and in extra-embryonic fluids over a period of organogenesis in the cynomolgus monkey is similar to the reduction detected in rodent embryos and fluids over a comparable developmental period, but the relative gradient between maternal blood pH and embryonic intracellular pH is different. The difference in the pH gradient between the two species may lead to differential transplacental distribution of exogenous and endogenous substances.

Localisation of glycoproteins and glycosaminoglycans during early eye development in the macaque.

The composition of the extracellular matrix (ECM) was examined in the developing lens and optic cup (stages 11-16) of the long-tailed monkey (Macaca fascicularis) using peroxidase immunocytochemistry. The glycoproteins, fibronectin, laminin, and collagen types I and IV, were consistently associated with basement membranes (BM) of ocular epithelia at all stages examined. Discontinuity of the optic cup BM was observed during the early stages of evagination (stages 11 and 12); the even distribution of all 4 components was reestablished by stage 13 when the optic vesicle is closely apposed to the thickened lens placode. While fibronectin was most predominant in the mesenchymal matrix, all 4 glycoproteins were observed to variable degrees in the periocular mesenchyme. Particularly strong glycoprotein reactivity was observed in the interspace between the invaginating lens vesicle and optic cup whereas no significant reactivity occurred within the lens, developing retina or future corneal epithelium. Two glycosaminoglycans, hyaluronic acid and chondroitin sulphate, had virtually identical widespread staining patterns in all ocular BM and throughout the periocular mesenchyme and adjacent epithelial tissues, including the lens and retina. The observed temporal and regional staining patterns suggest that these ECM components are morphogenetic factors in the macaque eye, facilitating the complex series of integrated tissue interactions, movements and shape changes during the earliest stages of lens and optic vesicle morphogenesis. The macaque offers a valuable model to study these interactions due to the prolonged period of ocular development which is morphologically identical to humans.

Assessment of the developmental toxicity of 2-ethylhexanoic acid in rats and rabbits.

This study was carried out to assess the developmental toxicity of orally administered 2-ethylhexanoic acid (2-EHA) throughout organogenesis in the rat and the rabbit. Treatment of Fischer 344 inbred rats with doses of 100 to 1000 mg 2-EHA/kg/day on (Gestation Days) (GD) 6-15 in a range-finding and a definitive study resulted in a high level of maternal death at 1000 mg/kg/day. Clinical signs of maternal toxicity, including increased liver weight, as well as increased resorptions, dead fetuses, and growth retardation, but no malformations, were observed at 500 mg/kg/day. Slight developmental toxicity, manifested as a reduction in skeletal ossification, occurred in fetuses exposed to 250 mg/kg/day. No adverse effects of treatment were associated with the lower 2-EHA doses (100 and 125 mg/kg/day). Maternal toxicity was also observed in range-finding and definitive studies in New Zealand white rabbits exposed to 25 to 1000 mg 2-EHA/kg/day on GD 6-18 with excessive mortality observed at the highest doses (500 and 1000 mg/kg/day). A low incidence of maternal death as well as abortion occurred following treatment with 125 and 250 mg 2-EHA/kg/day. Less severe clinical signs (reduced weight and food consumption and hypoactivity) were also observed in the 250 mg/kg/day group. There were no adverse effects on fetal viability, growth, or morphology at any dose level. Thus, exposure to 2-EHA during the entire period of organogenesis caused developmental toxicity only at maternally toxic doses in the rat or adverse maternal effects in the absence of developmental toxicity in the rabbit. No evidence of teratogenicity was associated with 2-EHA in this classical safety assessment regimen in either species. The no observed adverse effect levels (NOAELs) for maternal and developmental toxicities in rats are 250 and 100 mg/kg/day, respectively; the corresponding NOAELs for rabbits are 25 mg/kg/day (maternal) and > or = 250 mg/kg/day (developmental).