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.

An overview of the current challenges when using rabbits for prenatal developmental toxicity studies with consideration of the impact on data interpretation.

The rabbit prenatal developmental toxicity study is an international testing requirement for the identification and characterisation of the potential hazards of chemicals to human health. The importance of the rabbit for the detection of chemical teratogens is without question. However, the rabbit when used as a laboratory test species presents unique challenges affecting data interpretation. The purpose of this review is to identify the factors which may impact the behaviour of the pregnant rabbit and lead to significant inter-animal variability, confounding interpretation of maternal toxicity. Additionally, the importance of appropriate dose selection is discussed not least because of the conflicting guidance for identifying and defining acceptable maternal toxicity that lack reference to the rabbit in particular. The test guideline prenatal developmental toxicity study is often unable to distinguish between developmental effects as a consequence of maternal toxicity and those that are a direct effect of the test chemical on the offspring yet there is increasing pressure to use the highest possible dose levels to induce significant maternal toxicity which for the rabbit, a species little understood in toxicological terms and one that is highly susceptible to stress, is defined by very few endpoints. Interpretation of study data is further confounded by dose selection yet the developmental effects, even in the presence of maternal toxicity, are being used in Europe as the basis for classifying agents as reproductive hazards and the maternal effects are being used to define key reference values.

An automated technique for double staining of bone and cartilage in fetal mouse skeletal specimens using alizarin red S and Alcian blue.

Automated methods exist for double staining bone and cartilage in fetal rat, rabbit and mice using alizarin red S and Alcian blue. None of the published methods produce consistently stained bone and cartilage in mouse fetuses. Consequently, we modified an automated staining method for use with mouse fetuses. Methanol replaced 95% industrial denatured alcohol, and 5% glacial acetic acid was used instead of 20%. We describe here our new protocol, which has been used successfully to stain specimens for regulatory prenatal developmental toxicity studies.

Retinal folding in the term rabbit fetus-Developmental abnormality or fixation artifact?

UNLABELLED: Preclinical reproductive toxicology studies must be performed to provide information on the risk of causing fetal harm in pregnant patients. These studies detect fetal malformations, which may or may not be drug-related adverse events. In the rabbit fetus, "slight retinal folding" is commonly observed; there is anecdotal evidence that these folds may be caused by routine (Bouin's fluid) fixation. This study used magnetic resonance imaging (MRI) to assess rabbit retinal architecture in fresh specimens, which was then reassessed following Bouin's fluid fixation. A total of 30 fetuses from 5 litters were imaged. No retinal folding was detected in fresh specimens but it was observed in a majority of fetuses post-fixation. The use of Davidson's fixative followed by Bouin's fluid showed a markedly lower incidence of "slight retinal folding". CONCLUSION: slight retinal folds in the rabbit fetus are likely artifactual and can be reduced using Davidson's fixation prior to Bouin's.

A "good practice" approach to the quality and consistency of morphological examination of the internal head structures of the term rabbit fetus.

Variation in the interpretation of the regulatory guidelines has resulted in a diversity of techniques employed to examine the internal structures of the foetal rabbit head. Examination of the foetal rabbit brain, using a single transverse section as the sole technique, is considered not to be sufficiently thorough to be regarded as an adequate examination method. It is not compliant with published EPA and OECD guidelines covering required examination of the internal head structures, nor is it considered to conform to the spirit of the safety assessment required by the ICH guideline. Fixation of approximately half of the heads in each litter to allow the examination of multiple transverse sections enables the major structures within the head to be assessed effectively. This method is compliant with current guidelines, represents "good practice" and should be consistently adopted for the examination of the internal head structures of the term rabbit foetus.

Imaging and morphology in reproductive toxicology - progress to date and future directions.

This review looks at the recent development and application of imaging techniques for the morphological examination of fetuses from preclinical regulatory reproductive toxicology studies. Full replacement of the examination methods currently used in routine studies (microdissection, Bouin's fluid fixation/sectioning and alizarin red S/alcian blue preparations) by imaging techniques has yet to be achieved. Progress, especially in the application of micro-CT for skeletal examination, has been made but challenges, particularly the financial investment required, remain. Despite this apparent lack of progress the application of imaging techniques to "non-routine" preclinical reproductive toxicology studies has been used to good effect. The ability to acquire multiple images over a time course i.e. longitudinally has enabled the fate, particularly of skeletal features, to be determined. The additional evidence gained from such studies can be used to better inform the prenatal developmental hazard assessment of test compounds.

Soft tissue examination of the fetal rat and rabbit head by magnetic resonance imaging.

The use of magnetic resonance imaging of the fetal rat and rabbit head, as an alternative to the traditional methods of fixation and preparation of serial sections, is described. Labeled magnetic resonance images of normal head anatomy have been provided as a reference for use when evaluating the internal structures of the head.

Use of magnetic resonance imaging (MRI) and micro-computed tomography (micro-CT) in the morphological examination of rat and rabbit fetuses from embryo-fetal development studies.

Images of the fetal skeleton and soft tissues of the head can be acquired using micro-CT and MRI respectively. Preliminary work has shown that the image acquisition times of commercially available micro-CT and MRI instruments are now sufficiently short, whilst still providing adequate image resolution, to allow high quality imaging of fetuses from embryo-fetal development (EFD) studies. Bespoke fetus holders, which allow the imaging of multiple specimens in a single imaging "run", have been used to increase throughput. Protocols have been devised that incorporate these technologies into routine rat and rabbit fetal examination regimes. It is intended to undertake evaluations of these protocols, using number of fetuses that replicate those that would be expected from normal EFD studies. Incorporation of these technologies is anticipated to allow all soft tissue and skeletal examination data to be collected on the same day, markedly reducing the time taken to provide data for evaluation.