Luc De Schaepdrijver

Zebrafish developmental toxicity assay: A fishy solution to reproductive toxicity screening, or just a red herring?

The zebrafish embryotoxicity/teratogenicity assay is described as a useful alternative screening model to evaluate the effect of drugs on embryofoetal development. Fertilized eggs were exposed to different concentrations of 15 compounds with teratogenic (8) and non-teratogenic (7) potential until 96h post-fertilization when 28 morphological endpoints and the level of compound uptake was assessed. The majority of drugs testing positive in mammals was also positive in zebrafish (75% sensitivity), while a relative high number of false positives were noted (43% specificity). Compound uptake determination appears useful for clarifying classifications as teratogenic or potential overdose although assay sensitivity could be improved to 71% if the exposure threshold, previously suggested as ∼50ng/larvae, is reconsidered. The zebrafish assay shows some potential, though limited in its current form, as a screening tool for developmental toxicity within Janssen drug development. Further assay refinement with respect to endpoints and body burden threshold is required.

In vivo longitudinal micro-CT study of bent long limb bones in rat offspring.

Micro-computed X-ray tomography (micro-CT) has been reported as a reliable method to assess ex vivo rat and rabbit fetal skeletons in embryo-fetal developmental toxicity studies. Since micro-CT is a non-invasive imaging modality it has the potential for longitudinal, in vivo investigation of postnatal skeletal development. This is the first paper using micro-CT to assess the reversibility of drug-induced bent long bones in a longitudinal study from birth to early adulthood in rat offspring. Analysis of the scans obtained on postnatal Day 0, 7, 21 and 80 showed complete recovery or repair of the bent long limb bones (including the scapula) within the first 3 weeks. When assessing risk the ability to demonstrate recovery is highly advantageous when interpreting such transient skeletal change. In summary, in vivo micro-CT of small laboratory animals can aid in non-clinical safety assessment, particularly for specific mechanistic purposes or to address a particular concern in developmental biology.

Value of juvenile animal studies

The Developmental and Reproductive Toxicology Technical Committee of the ILSI Health and Environmental Sciences Institute has undertaken a project to address the impact of juvenile animal studies on pediatric drug development. A workshop, sponsored and organized by the Health and Environmental Sciences Institute Developmental and Reproductive Toxicity Technical Committee, was held on May 5-6, 2010, in Washington, DC, to discuss the outcome of a global survey and the value of juvenile animal studies in the development of drugs intended for use in pediatric patients. During this workshop, summary data from the 2009-2010 survey were presented, and breakout sessions were used to discuss specific case studies to try to assess the impact of juvenile animal studies performed to support specific pediatric drug development. The objectives of the Workshop on The Value of Juvenile Animal Studies were to (1) provide a forum for scientists representing industry, academia, and regulatory agencies to discuss the impact of juvenile animal studies on pediatric drug development, (2) evaluate summary data from the survey to understand how the juvenile study data are being used and their impact in labeling and risk assessment, (3) discuss selected case studies from the survey to highlight key findings, and (4) identify the areas of improvement for the designs of juvenile animal studies. The take home message that resonated from the workshop discussions was that well-designed juvenile animal studies have demonstrated value in support of certain pediatric drug development programs. However, it was also clear that a juvenile animal study is not always warranted.

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.

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.

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.

Folic acid and primary prevention of neural tube defects: A review

With 4 mg folic acid daily, it may take 20 weeks to reach red-blood-cell folate levels between 1050 and 1340 nmol/L, optimal for reduction of the neural tube defect risk. Therefore, folic acid supplementation should be started 5-6 months before conception. The residual risk with optimal red-blood-cell folate levels is reportedly 4.5 per 10,000 total births. The residual risk in pooled data from countries with mandatory folic acid fortification is 7.5 per 10,000 pregnancies, regardless of pre-fortification rates. European monitoring of folate intake with questionnaires should be replaced by periodic measurements of red-blood-cell folate. The risk of folate intake >1 mg/day does not outweigh the benefits of folic acid fortification, provided un-metabolized folic acid, RBC folate and vitamin B12 are monitored periodically. A European monitoring system, based on U.S. National Health and Nutrition Examination Surveys, should reside with the European Centre for Disease Prevention and Control.

Maternal Stress Affects Fetal Growth but Not Developmental Instability in Rabbits

Developmental instability (DI), often measured by fluctuating asymmetry (FA) or the frequency of phenodeviants (fPD), is thought to increase with stress. However, specifically for stressors of maternal origin, evidence of such negative associations with DI is scarce. Whereas effects of maternal stress on DI have predominately been examined retroactively in humans, very little is known from experiments with well-defined stress levels in animal model systems. The aim of this study was to examine the effects of maternal exposure to three doses (plus a control) of a toxic compound affecting maternal condition on DI of their offspring in rabbits. Presence of maternal stress induced by the treatment was confirmed by a decrease in food consumption and weight gain of gravid females in the medium and high dose. Major abnormalities and mortality were unaffected by dose, suggesting the lack of toxic effects of the compound on the offspring. In spite of string maternal stress, offspring FA did not increase with dose. The treatment did lead to elevated fPD, but most were transient, reflecting growth retardation. Furthermore, a consistent association between fPD and FA was absent. These findings indicate that DI is not increased by maternal stress in this animal model.

Juvenile animal testing of hydroxypropyl-β-cyclodextrin in support of pediatric drug development.

Hydroxypropyl-β-cyclodextrin (HP-β-CD) is being explored as excipient for administration of poorly soluble NCEs in pediatrics. In support of pharmaceutical development, non-clinical studies were performed to investigate whether oral and intravenous administration of HP-β-CD showed a different response in juvenile rats versus adult rats. Juvenile rats received HP-β-CD via the intravenous route at dose levels of 50, 200 and 400mg/kg/day from postnatal day 16 to 44, or via oral gavage at 500, 1000 and 2000mg/kg/day from postnatal day 4 to 46. In addition to in vivo parameters, toxicokinetics and post-mortem evaluations were conducted. The main findings were related to the renal excretion of intact HP-β-CD and were regarded as non-adverse transient adaptive responses. The pathogenesis of the osmotic nephrosis-like changes are discussed. With increasing age a more effective renal clearance of HP-β-CD is present in line with the postnatal functional maturation of the kidney. In addition, following oral administration an increase in soft stools was seen which was related to osmotic water retention in the large intestine. The findings in the juvenile studies are very similar to those observed in previously performed adult rat studies at similar dose levels, same routes and similar or longer dose duration. No novel toxicity was seen in the juvenile studies.