Micro-CT imaging: Developing criteria for examining fetal skeletons in regulatory developmental toxicology studies - A workshop report.

During the past two decades the use and refinements of imaging modalities have markedly increased making it possible to image embryos and fetuses used in pivotal nonclinical studies submitted to regulatory agencies. Implementing these technologies into the Good Laboratory Practice environment requires rigorous testing, validation, and documentation to ensure the reproducibility of data. A workshop on current practices and regulatory requirements was held with the goal of defining minimal criteria for the proper implementation of these technologies and subsequent submission to regulatory agencies. Micro-computed tomography (micro-CT) is especially well suited for high-throughput evaluations, and is gaining popularity to evaluate fetal skeletons to assess the potential developmental toxicity of test agents. This workshop was convened to help scientists in the developmental toxicology field understand and apply micro-CT technology to nonclinical toxicology studies and facilitate the regulatory acceptance of imaging data. Presentations and workshop discussions covered: (1) principles of micro-CT fetal imaging; (2) concordance of findings with conventional skeletal evaluations; and (3) regulatory requirements for validating the system. Establishing these requirements for micro-CT examination can provide a path forward for laboratories considering implementing this technology and provide regulatory agencies with a basis to consider the acceptability of data generated via this technology.

Carcinogenicity and hormone studies with the tissue-selective estrogen receptor modulator bazadoxifene.

Bazedoxifene Acetate (BZA) is a selective estrogen receptor modulator (SERM) that is approved for the prevention and/or treatment of osteoporosis in postmenopausal women. To assess for carcinogenic potential, BZA was administered ad libitum in the diet to rats for 2 years. BZA caused an increase in benign ovarian tumors in female rats and decreased incidences of mammary tumors (females) and pituitary tumors (males and females). In addition, BZA provided a significant survival benefit at all dosages tested, which correlated with a significant reduction in pituitary and mammary gland tumors and decreased body weight gain (both genders). Additional studies were subsequently conducted in rats and monkeys to further explore the mechanisms likely responsible for the observed effects. Results from studies in hypophysectomized and chemically castrated female rats indicated that BZA did not directly stimulate formation of ovarian cysts, but an intact pituitary was required for cyst formation. Further, BZA increased estradiol concentrations in rats and monkeys. In monkeys, BZA increased concentrations of luteinizing hormone (LH) after onset of treatment and prohibited the preovulatory surge of LH until after cessation of treatment. These hormonal changes suggest that BZA inhibited both the positive and negative feedback effects of estrogen on gonadotropins and the resulting increase in LH caused formation and persistence of ovarian cysts, which eventually transformed into benign ovarian granulosa cell tumors in the rat carcinogenicity study. These results also suggest that the reductions in pituitary and mammary gland tumors were attributed to BZA-related antagonism of endogenous estrogens at the estrogen receptors.

Reproductive performance and early postnatal development in interleukin (IL)-13-deficient mice.

OBJECTIVE: The purpose of this study was to assess the effect of interleukin (IL)-13 deficiency on fertility and reproductive performance of adult mice and on morphological and behavioral development of the offspring. METHODS: Wild-type and homozygous IL-13-deficient (KO) mice were grouped by genotype, and male and female mice were mated within each group. Adult (F(0) ) mice were evaluated for reproductive performance, and development was assessed in F(1) fetuses on gestation day 18, and in F(1) pups to postnatal day 35. RESULTS: In F(0) males, there were no differences in the number of males that mated or impregnated females, or in total sperm count or sperm motility, between the wild-type and KO groups. In F(0) females, there were no observed genotype-related differences in fertility, length of gestation, number of viable fetuses per litter, or viability of offspring. There were no differences in embryo-fetal development (external/palate, skeletal, visceral) of the F(1) fetuses between genotypes. Similarly, IL-13 deficiency had no impact on any postnatal parameters assessed including reflex, sexual maturation, learning, and memory. CONCLUSIONS: IL-13 deficiency had no observed effect on reproductive performance or morphological and behavioral development in mice.

Assessment of immunization against recombinant human bone morphogenetic protein-2 (dibotermin alpha) on reproduction and development in rabbits.

BACKGROUND: To determine if the fetus was affected by maternal antibodies to BMP-2, the antibody response and developmental effects in fetuses from does immunized against recombinant human BMP-2 were evaluated. METHODS: Female New Zealand White rabbits received four intramuscular injections (on premating days 1, 8, 22, and 43 [3 days before mating]) of saline and adjuvant (TiterMax(®) Gold [control]) or recombinant human BMP-2 (2 mg/dose) and adjuvant (treated). On GD 29, fetuses were examined, and maternal and fetal anti-BMP-2 titer levels and neutralizing activity were assessed. RESULTS: Anti-BMP-2 antibodies were detected in 17 of 18 treated does (127 of 151 fetuses), and low levels were detected in 2 of 16 control does (no fetal exposure observed). In general, levels of fetal anti-BMP-2 antibodies were similar to those in the does, and pregnancy did not boost the immune response to BMP-2. There were no effects of immunization or anti-BMP-2 antibody titer levels on embryo-fetal viability, fetal weight, or fetal external, visceral, or skeletal development. Only a small number of fetuses (n = 4) displayed detectable neutralizing anti-BMP-2 antibodies, but there were no treatment-related effects in those fetuses. CONCLUSIONS: The lack of embryo-fetal effects may be due to dosage effects of neutralizing anti-BMP-2 antibodies, timing of exposure (stage and duration) to neutralizing anti-BMP-2 antibodies, and/or redundancy of effects of the various BMPs.