L. David Wise

Placental P-glycoprotein deficiency enhances susceptibility to chemically induced birth defects in mice

A subpopulation of the CF-1 mouse strain contains a spontaneous mutation in the P-glycoprotein (Pgp) mdr1a gene, which leads to a lack of mdr1a expression in the placenta as well as brain and intestine. Individual CF-1 mice can be identified according to their Pgp status by a restriction fragment length polymorphism. Male and female mice selected on the basis of Pgp genotype were mated and the pregnant dams exposed during gestation to the known Pgp substrate, L-652,280, the 8,9 Z photoisomer of the naturally occurring avermectin Bla, which is known to produce cleft palate in mice. Fetal examination demonstrated that within individual litters, fetuses deficient in Pgp (-/-) were 100% susceptible to cleft palate, whereas their +/- heterozygote littermates were less sensitive. The homozygous +/+ fetuses with abundant Pgp were totally insensitive at the doses tested. The degree of chemical exposure of fetuses within each litter was inversely related to expression of placental Pgp, which was determined by the fetal genotype. These results demonstrate the importance of placental Pgp in protecting the fetus from potential teratogens and suggest that Pgp inhibitors should be carefully evaluated for their potential to increase susceptibility to chemical-induced teratogenesis.

Methods for assessing sperm motility, morphology, and counts in the rat, rabbit, and dog: A consensus report☆

Reproductive toxicity studies are increasingly including assessments of sperm parameters including motility, morphology, and counts. While these assessments can provide valuable information for the determination of potential reproductive toxicity, the methods for conducting the assessments have not been well developed in all laboratories and are continually evolving. The use of different methods in different laboratories makes comparison of data among laboratories difficult. To address the differences in methods, a working group was convened to discuss methods currently in use, share data, and try to reach consensus about optimal methods for assessing sperm parameters in rats, rabbits, and dogs. This article presents the consensus report, as well as future research needs, with the hope that optimized common methods will aid in the detection of reproductive effects and enhance interlaboratory comparisons.

Terminology of developmental abnormalities in common laboratory mammals (version 1)

This paper presents the first version of an internationally-developed glossary of terms for structural developmental abnormalities in common laboratory animals. The glossary is put forward by the International Federation of Teratology Societies (IFTS) Committee on International Harmonization of Nomenclature in Developmental Toxicology, and represents considerable progress toward harmonization of terminology in this area. The purpose of this effort is to provide a common vocabulary that will reduce confusion and ambiguity in the description of developmental effects, particularly in submissions to regulatory agencies worldwide. The glossary contains a primary term or phrase, a definition of the abnormality, and notes, where appropriate. Selected synonyms or related terms, which reflect a similar or closely related concept, are noted. Nonpreferred terms are indicated where their usage may be incorrect. Modifying terms used repeatedly in the glossary (e.g., absent, branched) are listed and defined separately, instead of repeating their definitions for each observation. Syndrome names are generally excluded from the glossary, but are listed separately in an appendix. The glossary is organized into broad sections for external, visceral, and skeletal observations, then subdivided into regions, structures, or organs in a general overall head to tail sequence. Numbering is sequential, and not in any regional or hierarchical order. Uses and misuses of the glossary are discussed. Comments, questions, suggestions, and additions from practitioners in the field of developmental toxicology are welcomed on the organization of the glossary as well as on the specific terms and definitions. Updates of the glossary are planned based on the comments received.

Developmental neurotoxicity evaluation of acrylamide in Sprague-Dawley rats

Based on the literature to-date, the potential of acrylamide (ACRL) to cause developmental neurotoxicity in laboratory animals has not been assessed. We examined this potential in Sprague-Dawley rats using a study design similar to that proposed by the USEPA. Dosages of 0 (deionized water), 5, 10, 15, or 20 mg/kg/day were administered at 5 ml/kg by oral gavage from gestational day 6 to lactational day 10 to groups of 12 mated females each. Females were allowed to deliver and the offspring were evaluated for survival, growth, development, behavior, and histological changes to brain, spinal cord, and peripheral nerve. Behavioral assessments consisted of open-field motor activity, auditory startle habituation, and passive avoidance tests during both the preweaning and adult periods (1 animal/sex/litter). All F0 and F1 animals in the 20 mg/kg/day group were euthanized early in the lactation period due to high pup mortality. Significantly increased pup mortality was also present in the 15 mg/kg/day group. There were dose-related decreases in average F0 maternal body weight gains during the dosing period in the 10, 15, and 20 mg/kg/day groups, and characteristic hindlimb splaying was observed in dams of the two highest dosage groups. Pup body weight proved to be the most sensitive indicator of developmental toxicity. Dose-related decrease in preweaning average weights were observed at all dose levels, although only transiently in the 5 mg/kg/day group. Average weight gain during the postweaning period was significantly decreased only in males of the 15 mg/kg/day group. Significant decreases in average horizontal motor activity and auditory startle response were observed only in weanlings of the 15 mg/kg/day group. The only behavioral effect in F1 adult animals was a decrease in auditory startle response in females of the 15 mg/kg/day group. There were no effects in the passive avoidance test or in the histological examination of the nervous system of preweaning pup or adult animals. Based on these results, the NOAEL (No Observed Adverse Effect Level) for developmental toxicity is less than 5 mg/kg/day, the NOAEL for maternal toxicity is 5 mg/kg/day, and that for developmental neurotoxicity is 10 mg/kg/day. Thus behavioral changes in the offspring were observed only at a dose which was also maternally toxic. These results suggest that acrylamide may be a selective developmental toxicant but not a selective developmental neurotoxicant, because a conventional measure of offspring toxicity (i.e., pup body weight) was affected at a dosage lower than that which produced maternal effects or offspring behavioral effects.

High-throughput micro-computed tomography imaging as a method to evaluate rat and rabbit fetal skeletal abnormalities for developmental toxicity studies

INTRODUCTION Fetal skeletal assessments are routinely conducted as a part of preclinical safety studies to support the development of novel therapeutic agents. Alizarin red staining with visual inspection of fetal skeletons is the gold standard in evaluating skeletons for the presence of developmental abnormalities. X-ray based micro-computed tomography (micro-CT) imaging has been used to evaluate small skeletal structures, both in vivo and ex vivo. Recent technological advances have reduced micro-CT image acquisition time making this technology practical for routine fetal skeletal evaluations. Herein we report on the use of micro-CT imaging as a method to perform high-throughput assessment of fetal skeletons. METHODS Micro-CT imaging of rat and rabbit fetal skeletons was conducted under a variety of conditions, including, in vivo, contrast-enhanced in vivo, and ex vivo. To increase throughput, micro-CT imaging was employed using custom designed polystyrene foam fetal holders to image entire litters of ex vivo fetuses. After micro-CT imaging, fetuses were routinely stained with alizarin red to compare micro-CT imaging results with traditional alizarin red staining. RESULTS Fetal skeletons could be visualized using in vivo micro-CT imaging; however, due to crowding, specific identification of individual fetuses was deemed not practical. Administration of a routine contrast agent to pregnant females highlighted maternal vascular structures including the placenta, but unfortunately, did not cross the placenta and did not highlight any fetal soft tissue structures. Ex vivo fetal imaging provided the best image quality of fetal skeletons and allowed for specific fetal identification. The fetal holders allowed for micro-CT imaging of approximately 400 rat fetuses or approximately 140 rabbit fetuses per hour. Micro-CT image skeletal findings and alizarin red findings were comparable. The very few discrepancies between the two methods involved the smallest skeletal elements with minimal ossification. DISCUSSION In conclusion, micro-CT ex vivo imaging can provide a reliable high-throughput method to assess fetal skeletal abnormalities for developmental toxicity studies.

Pre- and postnatal developmental toxicity study design for pharmaceuticals.

Assessment of potential developmental and reproductive toxicity of human pharmaceuticals is currently guided by the ICH S5(R2) document. The studies that assess the hazard of both pre- and postnatal exposure are predominantly conducted in rodents (rat and mouse). Utilizing the collective experience of the authors, acceptable designs for both the range-finding and definitive studies are presented with detailed descriptions for the presentation of data. In addition, the suggested initiation and then total duration of these studies in relation to clinical studies are described. Optional parameters that may be included in the studies, as well as possible combination with other study designs are discussed. The details described herein will assist all laboratories performing these studies, individuals who need to plan for the studies, and regulatory agencies that ultimately review these studies.

Comparison of motility and membrane integrity to assess rat sperm viability.

Rat sperm motility and membrane integrity were compared as endpoints for viability. Sperm motility was measured by computer-assisted semen analysis (CASA), whereas membrane integrity was assessed by flow cytometric analysis of sperm stained with two nucleic acid stains, SYBR-14 and propidium iodide. The two techniques were compared in experiments that examined sperm viability over time and by analysis of known mixtures of control and freeze/thaw-killed sperm. Results from the two approaches were quantitatively very similar. Sperm from rats treated with dibromoacetic acid (600 or 1200 mg/kg) or alpha-chlorhyrin (100 mg/kg) were also analyzed. Neither technique detected a treatment-related effect with dibromoacetic acid. CASA identified a significant decrease in sperm motility in alpha-chlorhyrin-treated rats, whereas flow cytometric analysis did not find a measureable change in sperm membrane integrity. Because decreases in sperm motility would be expected to directly affect fertility, CASA may be a more robust endpoint for risk assessment in reproductive toxicology studies than flow cytometric analysis of membrane integrity.

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.

Reversible decreases of fertility in male Sprague-Dawley rats treated orally with finasteride, a 5α-reductase inhibitor

Finasteride, a 5 alpha-reductase inhibitor, was investigated for its effects on fertility in male rats as part of its preclinical safety assessment. Studies were initiated when the male Sprague-Dawley rats were either young (4 to 6 weeks old) or mature (15 weeks old). Treatment duration ranged from 6 to 32 weeks. Each male was cohabited with two untreated females at various periods during and after treatment. Litter parameters were evaluated on either day 14 or 20 of gestation. Males were necropsied at the end of treatment or 7 to 11 weeks following the end of treatment. The major findings of these studies were that 1) young rats given 20 to 80 mg/kg/day of finasteride first showed mild to moderate decreases in fertility after 12 weeks of treatment, whereas mature males (given only 80 mg/kg/day) did not show a similar decrease until 24 weeks of treatment, 2) fewer copulatory plugs and atrophy of prostates and seminal vesicles were associated with finasteride treatment, 3) the decreased fertility was only partial (ie, fertility index did not decrease below 48% of control in any study) and was not due to decreases in mating, 4) formation of copulatory plugs, organ weights, and fertility returned to normal levels after at least 6 weeks of drug withdrawal, and 5) the testes showed no histologic or weight changes that would explain the effect on fertility. These results show that the decreased fertility in male rats was associated with finasteride-induced inhibition of accessory gland secretions, an expected pharmacologic effect.

Evaluation of hydroxyurea-induced fetal skeletal changes in Dutch belted rabbits by micro-computed tomography and alizarin red staining.

BACKGROUND This laboratory has been investigating the utility of X-ray micro-computed tomography (micro-CT) to produce high-resolution, 3D images of skeletal structures in common laboratory species. The present investigation uses micro-CT evaluation of skeletons from rabbit fetuses exposed to the known teratogen, hydroxyurea. METHODS Groups of 4-6 mated Dutch Belted female rabbits each were administered vehicle or hydroxyurea (62.5 to 500 mg/kg) once on GD 12. On GD 28, all live fetuses were weighed, euthanized, and viscera removed. Up to 7 fetuses per litter were placed into a custom-made polystyrene holder and scanned in the micro-CT imaging system. Raw projection data were acquired in approximately 15 seconds, and reconstructed images at 100-micron cubic voxel dimension could be viewed as early as 20 minutes later. Fetuses were subsequently stained with alizarin red, and findings recorded separately for each method without knowledge of treatment group. RESULTS Except for a few isolated cases, micro-CT evaluation detected the same skeletal malformations, variations, and incomplete ossifications as seen by the staining method. Skeletal elements that are very small (e.g., caudal-most vertebrae, metacarpal no. 1) or those with a minimal degree of ossification were occasionally not observed with micro-CT. However, this difference did not impact the overall study conclusions. Femur length was easily measured by micro-CT. CONCLUSIONS These results indicate that micro-CT imaging can effectively assess rabbit fetal skeletal structures, and for those laboratories with this resource, may be used to significantly reduce time prior to skeletal evaluation and hazardous wastes associated with staining.