A Probability Analysis of Historical Pregnancy and Fetal Data from Dutch Belted and New Zealand White Rabbit Strains from Embryo-Fetal Development Studies.

Embryo-fetal development (EFD) studies, typically in pregnant rats and rabbits, are conducted prior to enrolling females of reproductive age in clinical trials. Common rabbit strains used are the New Zealand White (NZW) and Dutch Belted (DB). As fetal abnormalities can occur in all groups, including controls, Historical Control Data (HCD) is compiled using data from control groups of EFD studies, and is used along with each study's concurrent control group to help determine whether fetal abnormalities are caused by the test article or are part of background incidences. A probability analysis was conducted on 2014 HCD collected at Charles River Inc., Horsham PA on Covance NZW, Covance DB, and Charles River (CR) NZW rabbits. The analysis was designed to determine the probability of 2 or 3 out of a group of 22 does aborting their litter or of having a fetal abnormality by chance. Results demonstrate that pregnancy parameters and fetal observations differ not only between strains, but between sources of rabbits of the same strain. As a result the probability of these observations occurring by chance in two or three litters was drastically different. Although no one single strain is perfect, this analysis highlights the need to appreciate the inherent differences in pregnancy and fetal abnormalities between strains, and points out that an apparent isolated increased incidence of an observation in one strain will not necessarily be test-article related in another strain. A robust HCD is critical for interpretation of EFD rabbit studies, regardless of the rabbit strain used.

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.

Comparison of a modified mid-coronal sectioning technique and Wilson's technique when conducting eye and brain examinations in rabbit teratology studies.

BACKGROUND: There are two methods used when examining fetal rabbit eyes and brain in teratology studies. One method employs prior fixation before serial sectioning (Wilson's technique) and the other uses fresh tissue (mid-coronal sectioning). METHODS: We modified the mid-coronal sectioning technique to include removal of eyes and brain for closer examination and to increase the number of structures that can be evaluated and compared it to the Wilson's technique. We found that external examination of the head, in conjunction with either sectioning method, is equally sensitive in identifying developmental defects. We evaluated 40,401 New Zealand White (NZW) and Dutch-Belted (DB) rabbit fetuses for external head alterations, of which 28,538 fetuses were further examined for eye and brain alterations using the modified mid-coronal sectioning method (16,675 fetuses) or Wilson's technique (11,863 fetuses). The fetuses were from vehicle control or drug-treated pregnant rabbits in embryo-fetal development studies conducted to meet international regulatory requirements for the development of new drugs. RESULTS: Both methods detected the more common alterations (microphthalmia and dilated lateral cerebral ventricles) and other less common findings (changes in size and/or shape of eye and brain structures). CONCLUSIONS: While both methods are equally sensitive at detecting common and rare developmental defects, the modified mid-coronal sectioning technique eliminates the use of chemicals and concomitant fixation artifacts that occur with the Wilson's technique and allows for examination of 100% intact fetuses thereby increasing potential for detecting eye and brain alterations as these findings occur infrequently in rabbits.

Concordance between alizarin red stained skeleton and micro-CT skeleton evaluation methods: A case study in New Zealand White rabbits.

OBJECTIVES: The objective of this study was to examine the fetal skeletons using both alizarin red stain and micro-computed tomography (CT) images; investigate differences, and to determine if the conclusions of the study were the same regardless of the examination method. METHODS: A candidate drug was given orally by gavage to pregnant New Zealand White rabbits on gestation day (GD) 7 to GD 19 (mating = GD 0) at doses of 0 (control), 0.02, 0.5, 5, and 15 mg/kg/day. Maternal toxicity was evident at ≥0.02 mg/kg/day. The 199 fetal skeletons (totaling 50,546 skeletal elements) obtained at cesarean delivery on GD29 were first stained with Alizarin Red S, then imaged by a Siemens Inveon micro-CT scanner. All fetal skeletons were examined by both methods, without knowledge of dose group, and the results were compared. RESULTS: In total, 33 types of skeletal abnormalities were identified. There was 99.8% concordance of results comparing stain to micro-CT. Ossification of the middle phalanx of the forepaw digit 5 showed the greatest difference between the two methods. CONCLUSION: Overall, micro-CT imaging is a realistic, and robust alternative to skeletal staining to examine fetal rabbit skeletons in developmental toxicity studies.

The potential role for corticosterone in the induction of cleft palate in mice after treatment with a selective NK-1 receptor antagonist, casopitant (GW679769B).

BACKGROUND: Casopitant is a potent and selective NK-1 receptor antagonist that has shown clinical efficacy in the prevention of chemotherapy-induced and postoperative-induced nausea and vomiting. METHODS: In an embryo-fetal development study, pregnant mice were given vehicle (sterile water) or doses of 30, 100, or 300 mg/kg/day casopitant on Gestation Day (GD) 6 to 15. Fetuses were evaluated for external, visceral, and skeletal abnormalities on GD 18. In a follow-on study to investigate casopitant-induced hormonal changes during the developmental period for palate formation, pregnant mice were given vehicle (sterile water) or 300 mg/kg/day casopitant once daily on GD 6 to 13. Blood was collected on GD 13 at various time-points for measurement of plasma adrenocorticotropic hormone and corticosterone (CRT) concentrations. RESULTS: There was no evidence of developmental toxicity in mice at 30 or 100 mg/kg/day but 9% of fetuses at 300 mg/kg/day had cleft palate. Mice are sensitive to glucocorticoid-induced cleft palates, and NK-1 antagonists are known to modulate the hypothalamic-pituitary-adrenal axis leading to increases in corticosterone. On GD 13, mean plasma adrenocorticotropic hormone levels at 300 mg/kg/day were elevated by approximately twofold from vehicle-treated levels at 1 hr post-dose and mean plasma CRT levels were elevated by 3, 5, and 10-fold at 0.5, 1, and 2 hr post-dose, respectively. CONCLUSIONS: The increased level of CRT was in the range previously shown in the literature to cause cleft palates in mice and was likely the underlying mechanism behind casopitant-induced cleft palate in mice.

Absence of developmental and reproductive toxicity in animals exposed to dolutegravir.

The success of new antiretroviral medicines for HIV resulted in a change to guidelines of standard therapy where continuation of antiretroviral therapy is recommended to maintain the low viral load during pregnancy, thereby preventing transmission of the virus to the fetus. As a result, pregnancy related exposure to HIV medicines has increased. Understanding the safety of these medicines during pregnancy is of paramount importance to ensure health of mothers and their offspring; well-designed animal studies that evaluate the reproductive life cycle play a key role in this effort. As part of the medicine development program for dolutegravir (DTG), a series of reproductive and developmental toxicity studies were conducted using rats and rabbits. In a fertility study, where exposure to DTG occurred in female rats before mating through conception and up to implantation of the embryo, no effects on reproductive cycles, ovulation, fertility) or preimplantation embryonic growth were observed. In rat and rabbit embryo-fetal development studies, where exposure to DTG occurred during organogenesis, no malformations or other developmental abnormalities were observed. In a rat pre- and post-natal development study, where DTG exposure to the pups occurred during pregnancy and postnatally via milk, no malformations or other developmental abnormalities were observed. In these studies, no DTG-related effects occurred on fertility, embryonic (pre- and post-implantation loss, resorptions, abortions, and malformations) or fetal development where the multiples of exposure at the maximum recommended human dose were up to 27 times higher in rats or below the human exposure in rabbits.

Terminology of developmental abnormalities in common laboratory mammals (Version 2).

This update (Version 2) of the Terminology of Developmental Abnormalities in Common Laboratory Mammals (Version 1) by Wise et al. (1997) incorporates improvements and enhancements to both content and organization of the terminology, to enable greater flexibility in its application, while maintaining a consistent approach to the description of findings. The revisions are the result of an international collaboration among interested organizations, advised by individual experts and the outcomes of several workshops. The terminology remains organized into tables under the broad categories of external, visceral, and skeletal observations, following the manner in which data are typically collected and recorded in developmental toxicity studies. This arrangement of the tables, as well as other information provided in appendices, is intended to facilitate the process of specimen evaluation at the laboratory bench level. Only the commonly used laboratory mammals (i.e., rats, mice, rabbits) are addressed in the current terminology tables. The inclusion of other species that are used in developmental toxicity testing, such as primates, is considered outside the scope of the present update. Similarly, categorization of findings as, for example, "malformation" or "variation" remains unaddressed, in accordance with the overall principle that the focus of this document is descriptive terminology and not diagnosis/interpretation. The skeletal terms have been augmented to accommodate cartilage findings.

The use of micro-CT imaging to examine and illustrate fetal skeletal abnormalities in Dutch Belted rabbits and to prove concordance with Alizarin Red stained skeletal examination.

OBJECTIVES: In our laboratory we evaluated the use of micro-computed tomography (micro-CT) using a high resolution acquisition protocol and fetuses obtained on Gestation Day (GD) 29 (mating = GD 0). METHODS: To show concordance between traditional Alizarin Red S stain and micro-CT skeletal examination methods, 103 fetuses from 19 untreated Dutch belted rabbits were obtained by cesarean section and stored frozen. The fetuses were thawed, imaged and examined digitally by micro-CT, then stained and re-examined using traditional methods. RESULTS: A total of 12 individual malformations and 35 unique variations were detected by both methods. Differences in the extent of ossification were found in only 51 of 26,196 bones while 99.8% of the observations were identical. Of the 51 differences, 31 were an unossified fifth medial phalanx of the forepaw indicating that very low-density skeletal bones may be visible by Alizarin Red stain but not by micro-CT scan. To establish this methodology under pharmaceutical testing conditions, we obtained and imaged by micro-CT Alizarin Red S stained abnormal fetal rabbit skeletons previously exposed to a drug candidate associated with craniofacial malformations in humans. All of the types of skeletal abnormalities first identified by staining were also detected by micro-CT examination. Representative images of these 66 different fetal skeletal abnormalities were characterized, and compiled to illustrate visual concordance between micro-CT scanned and traditional Alizarin Red S stained skeletons. CONCLUSION: Micro-CT imaging is an accurate, reliable and robust method that can be used as an alternative to stain when examining fetal rabbit skeletons in developmental toxicity studies.

Terminology of developmental abnormalities in common laboratory mammals (version 2).

This update (version 2) of the Terminology of developmental abnormalities in common laboratory mammals (version 1) by Wise et al. [Wise LD, Beck SL, Beltrame D, Beyer BK, Chahoud I, Clark RL, Clark R, Druga AM, Fueston MH, Guittin P, Henwood SM, Kimmel CA, Lindstrom P, Palmer AK, Petrere JA, Solomon HM, Yasuda M, York RG. Terminology of developmental abnormalities in common laboratory mammals (version 1). Teratology 1997;55:249-92] incorporates improvements and enhancements to both content and organization of the terminology, to enable greater flexibility in its application, while maintaining a consistent approach to the description of findings. The revisions are the result of an international collaboration among interested organizations, advised by individual experts and the outcomes of several workshops. The terminology remains organized into tables under the broad categories of external, visceral, and skeletal observations, following the manner in which data are typically collected and recorded in developmental toxicity studies. This arrangement of the tables, as well as other information provided in appendices, is intended to facilitate the process of specimen evaluation at the laboratory bench level. Only the commonly used laboratory mammals (i.e., rats, mice, rabbits) are addressed in the current terminology tables. The inclusion of other species that are used in developmental toxicity testing, such as primates, is considered outside the scope of the present update. Similarly, categorization of findings as, for example, "malformation" or "variation" remains unaddressed, in accordance with the overall principle that the focus of this document is descriptive terminology and not diagnosis/interpretation. The skeletal terms have been augmented to accommodate cartilage findings.

Terminology of developmental abnormalities in common laboratory mammals (version 2).

This update (Version 2) of the Terminology of Developmental Abnormalities in Common Laboratory Mammals (Version 1) incorporates improvements and enhancements to both content and organization of the terminology to enable greater flexibility in its application, while maintaining a consistent approach to the description of findings. The revisions are the result of an international collaboration among interested organizations, advised by individual experts and the outcomes of several workshops. The terminology remains organized into tables under the broad categories of external, visceral, and skeletal observations, following the manner in which data are typically collected and recorded in developmental toxicity studies. This arrangement of the tables, as well as other information provided in appendices, is intended to facilitate the process of specimen evaluation at the laboratory bench level. Only the commonly used laboratory mammals (i.e. rats, mice, rabbits) are addressed in the current terminology tables. The inclusion of other species that are used in developmental toxicity testing, such as primates, is considered outside the scope of the present update. Similarly, categorization of findings as, for example, 'malformation' or 'variation' remains unaddressed, in accordance with the overall principle that the focus of this document is descriptive terminology and not diagnosis or interpretation. The skeletal terms have been augmented to accommodate cartilage findings.

SB-236057: Critical window of sensitivity study and embryopathy of a potent musculoskeletal teratogen.

BACKGROUND: SB-236057 is a potent skeletal teratogen in rodents and rabbits. The study objective was to identify the critical developmental window of compound sensitivity and to characterize the early onset of SB-236057 embryopathy. METHODS: SB-236057 was orally administered to Sprague Dawley dams at 100 mg/kg/day on days 6-7, 8-11, 12-14, or 15-17 postcoitus (pc). The critical window of sensitivity was identified to occur between days 8-11 pc. Dams were then dosed on days 8-11 pc and embryos were evaluated by histochemical procedures on days 11, 13, or 15 pc. RESULTS: Axial malformations were evident by day 11 pc. Analysis of the cartilaginous skeleton revealed missing posterior axial skeletal elements. However, only about one-third of the malformed fetuses exhibited obvious rib and vertebrae abnormalities, and none of the affected fetuses exhibited abnormal appendicular skeletal elements. Expression pattern of sonic hedgehog in the notochord and floor plate was not affected, suggesting ventral midline signaling was not disrupted. Histological analysis demonstrated hypoplastic and/or missing musculature in proximity to the ribs and vertebrae. Caspase 3 analysis revealed no increases in apoptotic cells in the musculature. Confocal analysis of the limbs demonstrated truncated peripheral nerve formation and shortening of the appendicular musculature. CONCLUSIONS: SB-236057 is speculated to alter paraxial mesoderm programming. Many of the skeletal malformations may be caused secondarily from musculature abnormalities, suggesting that the myotome may be particularly sensitive to the compound. Furthermore, the finding that peripheral nerve trajectories were altered along the axis and in the limb suggests that SB-236057 may alter early embryonic signaling pathways necessary for neuronal differentiation/axonal guidance that occur subsequently in embryo-fetal development.