Belen Tornesi

Species-specificity of ethylene glycol-induced developmental toxicity: toxicokinetic and whole embryo culture studies in the rabbit

High-dose gavage exposure to ethylene glycol (EG) is teratogenic in rats, but not rabbits. To investigate the reason for this species difference, toxicokinetic and whole embryo culture (WEC) studies were conducted in gestation day 9 New Zealand White rabbits, and the data compared to very similar data previously generated in pregnant rats. In the toxicokinetic study, maximal levels of unchanged EG in rabbits were comparable to those reported for rats. However, maximal levels of EGs teratogenic metabolite, glycolic acid (GA), in rabbit maternal blood and embryo were only 46% and 10% of the respective levels in rats. The toxicokinetic profile suggested that the lower GA levels in rabbits were due to a slower rate of maternal metabolism of EG to GA, slow uptake of GA into the yolk sac cavity fluid which surrounds the embryo, and negligible transfer via the visceral yolk sac (VYS) placenta. In the WEC study, exposure of rabbit conceptuses to high concentrations (< or = 12.5 mM) of GA was without effect, which contrasts with reported effects in rat WEC at > or = 3 mM. Overall, these data implicate toxicokinetics as an important factor underlying the species difference, although intrinsic insensitivity of the rabbit embryo might also be involved. Integration of these findings with published human data suggest that the rabbit is the more relevant model for human EG exposure, based on the negligible role of the rabbit VYS in placental transfer (humans lack a VYS) and similar rates of EG metabolism and extraembryonic fluid turnover.

An in vitro screening paradigm for extracts of whole foods for detection of potential toxicants.

The application of organic, conventional and biotechnology techniques can alter the intrinsic levels of natural toxicants in crop foods and methods are needed to screen for unexpected changes in toxicant levels. We evaluated crude, aqueous preparations of 37 foods purchased from a local market in a battery of four in vitro mammalian toxicity screens. The foods were evaluated in one or more of the following tests: (1) cytotoxicity (37 foods) and (2) chromosomal aberration test (nine foods), both in Chinese hamster ovary cells, (3) limb bud micromass assay (nine foods) using 11-day old CD-1 mouse embryos and (4) estrogenicity (MCF-7 cells transfected with estrogen receptor and lucerifase reporter constructs, 12 foods). IC50s for cellular proliferation ranged from < 1% (v/v, garlic) to > 10% (v/v, 18 foods), the maximal concentration tested. Five of nine preparations (soybeans, broccoli, garlic, snow peas and corn) were clastogenic and two (soybeans and snow peas) inhibited chrondrogenesis in the limb bud micromass assay. Five of nine preparations (soybeans, snow peas, cumin, asparagus and bean sprouts) produced significant estrogenic responses. Overall, the 12 foods evaluated in two or more of the tests showed different patterns of response. These preliminary data indicate that screening for potential toxicants is possible with fast, relatively inexpensive in vitro tests. These in vitro tests, while potentially useful to detect unexpected toxicants in plants that may signal the need for further evaluation, are not directly useful to predict human or animal risk from eating these plants.

Comparing rat and rabbit embryo-fetal developmental toxicity data for 379 pharmaceuticals: on systemic dose and developmental effects.

Abstract A database of embryo-fetal developmental toxicity (EFDT) studies of 379 pharmaceutical compounds in rat and rabbit was analyzed for species differences based on toxicokinetic parameters of area under the curve (AUC) and maximum concentration (Cmax) at the developmental lowest adverse effect level (dLOAEL). For the vast majority of cases (83% based on AUC of n = 283), dLOAELs in rats and rabbits were within the same order of magnitude (less than 10-fold different) when compared based on available data on AUC and Cmax exposures. For 13.5% of the compounds the rabbit was more sensitive and for 3.5% of compounds the rat was more sensitive when compared based on AUC exposures. For 12% of the compounds the rabbit was more sensitive and for 1.3% of compounds the rat was more sensitive based on Cmax exposures. When evaluated based on human equivalent dose (HED) conversion using standard factors, the rat and rabbit were equally sensitive. The relative extent of embryo-fetal toxicity in the presence of maternal toxicity was not different between species. Overall effect severity incidences were distributed similarly in rat and rabbit studies. Individual rat and rabbit strains did not show a different general distribution of systemic exposure LOAELs as compared to all strains combined for each species. There were no apparent species differences in the occurrence of embryo-fetal variations. Based on power of detection and given differences in the nature of developmental effects between rat and rabbit study outcomes for individual compounds, EFDT studies in two species have added value over single studies.

Developmental and reproductive toxicology studies in IL‐12p40 knockout mice

BACKGROUND Interleukin (IL)-12 is a cytokine that can exert regulatory effects on T and NK cells. This study was designed to identify potential developmental and reproductive hazards associated with IL-12p40 knockout in mice. METHODS In the combined fertility and teratology study, female F0 C57/BL6 wild-type control mice and female F0 C57/BL6 IL-12p40 homozgyous knockout mice were assessed for estrous cyclicity, sperm, and mating parameters. Pregnant females were euthanized on gestation day (GD) 18 and their fetuses were assessed for external, visceral, and skeletal development. In the peri and postnatal development study, the F1 wild-type control and IL-12p40 knockout mice were assessed for developmental landmarks, sexual development, passive avoidance, motor activity, and morris water maze. RESULTS The IL-12p40 knockout male mice exhibited decreased testis weights when compared to the wild-type control group; however, this finding was not considered adverse, as it had no apparent functional effects on mating, fertility, and pregnancy rates or sperm motility. The IL-12p40 knockout group exhibited effects on estrous cycle length, passive avoidance, morris water maze, and motor activity when compared to the wild-type control group. However, since these findings were small in magnitude, transient and/or had no apparent effects on subsequent growth and development, they were not considered adverse. CONCLUSIONS These results demonstrate that although IL-12p40 homozygous knockout in mice exhibited effects on developmental and reproductive parameters, these effects were relatively minor and were not considered adverse.

Comparison of rat and rabbit embryo–fetal developmental toxicity data for 379 pharmaceuticals: on the nature and severity of developmental effects

Abstract Regulatory non-clinical safety testing of human pharmaceuticals typically requires embryo–fetal developmental toxicity (EFDT) testing in two species (one rodent and one non-rodent). The question has been raised whether under some conditions EFDT testing could be limited to one species, or whether the testing in a second species could be decided on a case-by-case basis. As part of a consortium initiative, we built and queried a database of 379 compounds with EFDT studies (in both rat and rabbit animal models) conducted for marketed and non-marketed pharmaceuticals for their potential for adverse developmental and maternal outcomes, including EFDT incidence and the nature and severity of adverse findings. Manifestation of EFDT in either one or both species was demonstrated for 282 compounds (74%). EFDT was detected in only one species (rat or rabbit) in almost a third (31%, 118 compounds), with 58% (68 compounds) of rat studies and 42% (50 compounds) of rabbit studies identifying an EFDT signal. For 24 compounds (6%), fetal malformations were observed in one species (rat or rabbit) in the absence of any EFDT in the second species. In general, growth retardation, fetal variations, and malformations were more prominent in the rat, whereas embryo–fetal death was observed more often in the rabbit. Discordance across species may be attributed to factors such as maternal toxicity, study design differences, pharmacokinetic differences, and pharmacologic relevance of species. The current analysis suggests that in general both species are equally sensitive on the basis of an overall EFDT LOAEL comparison, but selective EFDT toxicity in one species is not uncommon. Also, there appear to be species differences in the prevalence of various EFDT manifestations (i.e. embryo–fetal death, growth retardation, and dysmorphogenesis) between rat and rabbit, suggesting that the use of both species has a higher probability of detecting developmental toxicants than either one alone.

The Impact of Dose Rate on Ethylene Glycol Developmental Toxicity and Pharmacokinetics in Pregnant CD Rats

High-dose bolus exposure of rats to ethylene glycol (EG) causes developmental toxicity mediated by a metabolite, glycolic acid (GA), whose levels increase disproportionately when its metabolism is saturated. However, low-level exposures that do not saturate GA metabolism have a low potential for developmental effects. Toward the goal of developing EG risk assessments based on internal dose metrics, this study examined the differences between fast (bolus) and slow (continuous infusion) dose-rate exposures to EG on developmental outcome and pharmacokinetics. Time-mated female CD rats received sc bolus injections of 0, 1000, or 2000 mg/kg/day of EG on gestation day (GD) 6-15 once daily, whereas three corresponding groups were given the same daily doses as an infusion administered continuously from GD 6-15 via an sc implantable pump. In the sc bolus groups, increases in 11 fetal malformations (major defects) and 12 variations (minor alterations) were seen at the 2000 mg/kg/day dose level, whereas increases in 2 malformations and 2 variations occurred at 1000 mg/kg/day. In contrast, equivalent daily doses of EG given slowly via infusion did not cause any developmental effects. A pharmacokinetics time course was then conducted to compare GD 11-12 kinetics from oral bolus (gavage) exposure versus sc infusion of EG. Although dose rate had a modest impact (8- to 11-fold difference) on peak EG levels, peak levels of GA in maternal blood, kidney, embryo, and exocoelomic fluid were 59, 100, 49, and 56 times higher, respectively, following gavage versus the same dose given by infusion. These data illustrate how high-dose bolus exposure to EG causes a dramatic shift to nonlinear GA kinetics, an event which is highly unlikely to occur following exposures to humans associated with consumer and worker uses.

Developmental sensitivity to the induction of great vessel malformations by N-(2-aminoethyl)ethanolamine.

N-(2-Aminoethyl)ethanolamine (AEEA) induced malformations of the great vessels in the offspring of rats treated during gestation and early lactation (Schneider et al., 2012. Birth Defects Res B Dev Reprod Toxicol [in press]). The aim of this study was to determine if in utero exposure alone was sufficient to induce these malformations or whether a peri-postnatal exposure or physiological component was required. Three groups of five time-mated female Wistar Han rats were administered AEEA (250 mg/kg/day) by gavage from gestation day (GD) 6 to GD 19 (groups 1 and 2) or from GD 6 to postnatal day 3 (group 3). Animals were euthanized on GD 21 (group 1) or postnatal day 4 (groups 2 and 3), and the hearts of the offspring were examined for changes to the great vessels. The incidence of malformations in group 1 was 91.1%, and primarily consisted of high aortic arch and abnormal carotid course. One fetus had an aortic aneurysm. All fetuses in groups 2 and 3 were malformed, primarily exhibiting abnormal carotid course and aneurysms, which mainly affected the aorta, ductus arteriosus, and pulmonary trunk. The incidence of high aortic arch was lower relative to group 1. Aneurysms were more prevalent in group 3 compared to group 2. These findings indicate that exposure to AEEA during gestation alone was sufficient to induce malformations of the great vessels and aneurysms, which may be triggered by physiological changes that occur during or after birth, but that the critical period of susceptibility to AEEA-induced aneurysms in the rat extends beyond gestation into the early postnatal period.

Pre‐ and Postnatal Development in the Cynomolgus Monkey Following Administration of ABT‐874, a Human Anti‐IL‐12/23p40 Monoclonal Antibody

BACKGROUND ABT-874 is an anti-IL-12/23 monoclonal antibody that binds to the p40 subunit of human IL-12 and IL-23. As part of its preclinical safety assessment, studies were conducted to assess its potential effects on pre- and postnatal development in cynomolgus monkeys. METHODS In the embryo-fetal development studies, ABT-874 was administered once weekly subcutaneously to adult female cynomolgus monkeys at doses of 0, 5, 25, or 100 mg/kg during gestation days (GD) 20 to 48. Fetuses were examined for external, visceral, and skeletal development on GD 100 or 150. In the pre- and postnatal study, ABT-874 was administered once weekly subcutaneously to adult female cynomolgus monkeys at doses of 10, 50, or 200 mg/kg from GD 20 through postpartum day 182. Infants were examined from birth up to 9 months of age for morphological and functional development. Potential effects on the infant immune system were evaluated by immunophenotyping of peripheral blood lymphocytes and by T-dependent antibody response to KLH. RESULTS There was no ABT-874-related maternal toxicity or adverse effects on fetuses or infants. ABT-874 was present in maternal and fetal serum at GD 100 and 150, and in infant serum through day 63 postbirth. ABT-874 was also present at low levels in breast milk through postpartum day 175. CONCLUSIONS Exposure of cynomolgus monkey fetuses and infants to ABT-874 had no adverse effects on embryo-fetal or postnatal development.

Birth Control in Clinical Trials Industry Survey of Current Use Practices, Governance, and Monitoring

The Health and Environmental Sciences Institute (HESI) Developmental and Reproductive Toxicology Technical Committee sponsored a pharmaceutical industry survey on current industry practices for contraception use during clinical trials. The objectives of the survey were to improve our understanding of the current industry practices for contraception requirements in clinical trials, the governance processes set up to promote consistency and/or compliance with contraception requirements, and the effectiveness of current contraception practices in preventing pregnancies during clinical trials. Opportunities for improvements in current practices were also considered. The survey results from 12 pharmaceutical companies identified significant variability among companies with regard to contraception practices and governance during clinical trials. This variability was due primarily to differences in definitions, areas of scientific uncertainty or misunderstanding, and differences in company approaches to enrollment in clinical trials. The survey also revealed that few companies collected data in a manner that would allow a retrospective understanding of the reasons for failure of birth control during clinical trials. In this article, suggestions are made for topics where regulatory guidance or scientific publications could facilitate best practice. These include provisions for a pragmatic definition of women of childbearing potential, guidance on how animal data can influence the requirements for male and female birth control, evidence-based guidance on birth control and pregnancy testing regimes suitable for low- and high-risk situations, plus practical methods to ascertain the risk of drug-drug interactions with hormonal contraceptives.

The Inhibin B Response to Testicular Toxicants Ethylene Glycol Monomethyl Ether or Dibromoacetic Acid in Male Rats

BACKGROUND This study was conducted as part of an ILSI-HESIconsortium effort to assess the utility of circulating inhibin B as an early biomarker of testicular toxicity in rats. METHODS Two known testicular toxicants were selected for use in this study: ethylene glycol monomethyl ether (EGME) and dibromoacetic acid (DBAA). EGME (200 mg/kg/day), DBAA (250 mg/kg/day), or vehicle control (0.2% hydroxypropyl methylcellulose [HPMC]) was administered orally to male rats for 3, 6, or 14 consecutive days. On study days 4, 7, and 15, serum was collected for evaluation of inhibin B levels from all surviving animals and a subset of animals was necropsied from each of the control, EGME, and DBAAgroups. RESULTS Administration of EGMEresulted in spermatocyte degeneration in late stage tubules and spermatocyte depletion to stage III on day 4, progressing to loss of spermatocytes and round spermatids to stage VI by day 7 and continued germ cell loss and degeneration of elongating spermatids by day 15. Inhibin B levels among EGME-treated animals progressively decreased relative to their respective controls at all time points. Administration of DBAA was associated with spermatid retention at all three time points and abnormal residual bodies at days 7 and 15. Inhibin B levels among DBAA-treated animals decreased progressively relative to their respective controls on days 7 and 15. CONCLUSIONS Serum inhibin B levels in rats provided a signal of testicular toxicity for each of these known testicular toxicants administered at high levels; however, histopathology provided the earliest evidence of toxic effects.