Embryofetal development study of vismodegib, a hedgehog pathway inhibitor, in rats.

Vismodegib (Erivedge) is a first-in-class small-molecule hedgehog pathway inhibitor for the treatment of adults with advanced basal-cell carcinoma. Because this pathway is known to play key roles in patterning and growth during vertebrate development, vismodegib was anticipated to be embryotoxic. To support marketing applications, an embryofetal development study was completed in which a limited number of pregnant rats (n = 6/group) was administered vismodegib by oral gavage on gestation days 6 to 17. When vismodegib was administered at ≥60 mg/kg/day, doses associated with evidence of pharmacologic activity in previous rat toxicity studies, all conceptuses were resorbed at an early embryonic stage in the absence of significant maternal toxicity. When administered at 10 mg/kg/day, corresponding to an exposure (AUC0-24h ) approximately 15% of the median in patients at steady state, a variety of malformations were observed, including absent/fused digits in the hindlimb of multiple fetuses, multiple craniofacial abnormalities in one fetus, and an anorectal defect in one fetus. In addition, the incidence of variations, including dilated renal pelvis or ureter and incompletely or unossified skeletal elements, was significantly greater when compared with the controls. These results confirmed that vismodegib is likely to be embryotoxic at clinically relevant maternal exposures, and doses ≥60 mg/kg/day resulted in a 100% incidence of embryolethality that likely resulted from severe defects in early embryonic development. In contrast, craniofacial defects typically associated with hedgehog pathway inhibition were only observed in one fetus at the low dose of 10 mg/kg/day, which likely reflected minimal or intermittent pathway inhibition at low exposures.

The effects of interleukin-6 signal blockade on immune system, reproductive and skeletal development in juvenile mice.

Interleukin-6 (IL-6) is involved in the pathogenesis of multiple disorders, including juvenile autoimmune diseases. IL-6 participates in a broad spectrum of physiological events, and the IL-6 receptor (IL-6R) is widely distributed across multiple organs. The interrelationship of development phases in juveniles together with organs involved in IL-6 signaling called for evaluations of anti-IL-6R antibody induced effects in a juvenile mouse model to assess the safety of such an approach in human juvenile arthritis. Here we show that naive mice in which IL-6 signals have been transiently blocked during the juvenile period develop normally. The fatal immunogenic reactions recorded earlier by repeated administration of the chosen rat anti-mouse IL-6R antibody, MR16-1, to mice were avoided successfully by application of a high loading dose followed by lower maintenance doses, with the support of modeling data. The high loading-dose regimen enabled us to conduct assessments without any major interference due to immunogenicity. Transient and complete inhibition of IL-6 signals from postnatal days 22 to 79 in mice exhibited no biologically important changes in sexual maturation or development of immune and skeletal systems. Although tendencies toward reductions of peripheral blood T-cell counts were observed, normal levels of antigen-specific IgG/IgM antibody productions indicating sufficient immunological functions were confirmed. Our results demonstrate that blockage of IL-6R by the neutralizing antibody does not affect juvenile development. This may be in part due to the generation or existence of compensatory pathways in the whole body system.

Harmonization of description and classification of fetal observations: achievements and problems still unresolved: report of the 7th Workshop on the Terminology in Developmental Toxicology Berlin, 4-6 May 2011.

This article summarizes the 7th Workshop on the Terminology in Developmental Toxicology held in Berlin, May 4-6, 2011. The series of Berlin Workshops has been mainly concerned with the harmonization of terminology and classification of fetal anomalies in developmental toxicity studies. The main topics of the 7th Workshop were knowledge on the fate of anomalies after birth, use of Version 2 terminology for maternal-fetal observations and non-routinely used species, reclassification of "grey zone" anomalies and categorization of fetal observations for human health risk assessment. The paucity of data on health consequences of the postnatal permanence of fetal anomalies is relevant and further studies are needed. The Version 2 terminology is an important step forward and the terms listed in this glossary are considered also to be appropriate for most observations in non-routinely used species. Continuation of the Berlin Workshops was recommended. Topics suggested for the next Workshop were grouping of fetal observations for reporting and statistical analysis.

The effects of interleukin-6 signal blockade on fertility, embryo-fetal development, and immunization in vivo.

Possible effects of interleukin-6 (IL-6) on reproductive performance, embryonal development, parturition, and postnatal development have been suggested based on protein/mRNA expression level of IL-6 in related organs, but less is known about functions of IL-6 signals in these areas. Following two different approaches have been employed to investigate the role of IL-6 signals in fertility and pre-/postnatal development: administration of a rat anti-mouse IL-6 receptor antibody, MR16-1, to mice as a neutralizing antibody system, and B6.129S2-Il6(tm1Kopf)/J (IL-6 knockout [KO]) mice as a KO system. By intravenously dosing 50 mg/kg of MR16-1 every 3 days, animals in male and female fertility studies and dams in a pre-/postnatal development study exhibited plasma MR16-1 concentrations much higher than the effective plasma concentration, indicating that MR16-1 exposure was sufficient to completely block IL-6 signals. The concentration of MR16-1 in the plasma of fetuses exceeded that in the plasma of pregnant animals, and MR16-1 concentration in milk was about one-fourth of that in plasma. Both the transient IL-6 signal blockade by MR16-1, and the constitutive IL-6 signal inhibition using IL-6 KO mice in a combined fertility and pre-/postnatal development study, revealed no biologically important effects on fertility, early embryonic development to implantation, or pre-/postnatal development, including IgG/IgM production by keyhole limpet hemocyanin sensitization. These results indicate that IL-6 signals have no unique, noncompensable roles in reproduction and development in the whole body system, although contributions of IL-6 in the signaling network appear to exist, as suggested by previously published investigations.

Literature review on the genotoxicity, reproductive toxicity, and carcinogenicity of ethyl methanesulfonate.

In order to assess the risk of patients being exposed to an anti-AIDS medication contaminated with EMS we have performed in depth genotoxicity, general toxicity and DMPK investigations. The results of these studies are reported in the accompanying papers of this issue. Prior to starting our investigations we searched the literature for toxicity data on this well established mutagen with specific attention to dose-response relations in in vivo genotoxicity studies, since, obviously, in vivo data are pivotal for risk assessment. There are numerous published in vivo genotoxicity studies on EMS, with generally 50mg/kg - or higher - being the minimal dose used. The dose of 50mg/kg induced effects in some, but not all studies, while the dose of 100mg/kg was clearly positive in most studies, except for heritable mutations where a single dose of 100mg/kg was not observed to induce measurable effects in post-meiotic stages and even the maximal dose of 250 mg/kg was negative in pre-meiotic stages of male germ cell development. For somatic cells, NOEL values could not be derived for any of the endpoints studied. Although a large number of genotoxicity studies are available, none of the studies was sufficiently detailed to allow unambiguous conclusions about the presence of a (practical) threshold. But in most cases the dose-responses show a sublinear relationship (i.e. the slope increases with dose) which indicates that the data would not be incompatible with a threshold dose-response relationship. This stands in contrast to data on ethylnitrosourea (ENU) which has been studied concommittantly with EMS in several in vitro and in vivo genotoxicity investigations. ENU generally appeared to induce genotoxic effects with linear dose relationships. We also review the more limited data reported on teratogenicity and carcinogenicity of EMS. Induction of fetal malformations in mice appeared to have a NOEL of 100mg/kg. Classical life-time carcinogenicity studies have not been performed with EMS. Induction of mammary, lung, kidney, brain, and liver tumors has been observed after various short term treatment regimes. In none of the published studies a no effect level was reported and no exposure data are available. Overall, the experimental data do not fully characterize the carcinogenic potential of EMS and are insufficient for a risk extrapolation to humans. Although the data on teratogenicity and carcinogenicity are insufficient for assessing dose-response relations it is generally accepted that the genotoxic property of EMS is at the base of the teratogenic and carcinogenic effects.

The embryonic stem cell test for the early selection of pharmaceutical compounds.

Potential teratogenicity is a major consideration in the development of pharmaceutical substances. Currently its assessment involves large numbers of animal tests at high cost. This study assessed the feasibility of using the embryonic stem cell test (EST), validated by ECVAM in 1999, as a tool for the prediction of embryonic toxicity of pharmaceutical substances early in their development programmes. ESTs were carried out on 6 chemicals with well established toxicity characteristics established from literature and from the ECVAM study, and then on 10 Roche internal pharmaceutical substances already tested in vivo. The model correctly classified 81% of the substances. Further experiments are necessary to increase the database of the assay.

Harmonization of rat fetal external and visceral terminology and classification. Report of the Fourth Workshop on the Terminology in Developmental Toxicology, Berlin, 18-20 April 2002.

This article is a report on the Fourth Berlin Workshop on Terminology in Developmental Toxicology, which was held in April 2002. The workshop is part of an international project in the field of harmonization of terminology in developmental toxicology supported by IPCS. The goal of the Harmonization Project is to ensure better chemical risk assessment. The aim of this Fourth Workshop was to discuss the results of a previously conducted survey on classification of external and visceral anomalies, which are listed in the international glossary, developed under the auspices of IFTS (1997 glossary). The discussions among experts from research institutions, regulatory agencies, and industries were mainly focussed on terms for which there was disagreement and/or uncertainties and the possible reasons. For the illustration of "gray-zone" anomalies, pictures were provided by the participants, which constituted the basis for detailed discussions. There was high agreement that most of the external anomalies (>66%) should be classified as malformations. The few external anomalies for which there was low agreement to classify as a malformation were discussed in detail. None of the external findings, which had in the survey a high agreement, were categorized as a variation.A high agreement regarding the classification of approximately one-third of visceral anomalies was achieved with 34 and 2% being described as malformation and variation, respectively. Most of the visceral findings had low agreement indices and there appeared to be several reasons for this. Thus, the response, 'Not known/not used in the laboratory' (N) was often given. A couple of reasons for difficulties in the classification of an anomaly were that it is only rarely seen upon fetal examination or tends to be species specific. Furthermore, the classification of some anomalies as malformation or variation will remain vague as the decision must be made on a case-by-case basis. Factors affecting the decision include: the availability of appropriate historical control data, description of the grading and severity, whether the anomaly occurs in isolation or whether there is a relationship with an abnormal process, and finally, if the change represents an irreversible one, affecting human and/or animal health. It was concluded that a severity grading, supported by pictures of the anomaly, would be especially helpful to classify certain changes as malformation or as variation. Several of the soft tissue changes were considered likely to be the consequence of functional disorders and thus not strictly developmental anomalies. The possibility to describe a finding as 'Not Malformation' (Unclassified) was agreed upon. As a general conclusion it was emphasized that the observation of a permanent structural change should be considered to be a warning of possible consequences to humans, even when there is no apparent adverse effect on health and survival in adult animals of the species under investigation. Therefore, research is needed to further investigate postnatal consequences. Future collaboration in the field of reproductive and developmental toxicology should aim to further develop and implement a harmonized approach to the interpretation of study data. Therefore, this terminology work will continue in close cooperation with the IPCS Harmonization Project. A Steering Group should be established to facilitate the implementation of harmonized terminology into daily scientific work and its regulatory application.