A roadmap towards a human-centric safety assessment of advanced therapy medicinal products.

Advanced therapy medicinal products (ATMPs) are among the most complex pharmaceuticals with high human specificity. Species differences severely limit the clinical relevance of in vivo data. We conducted interviews with stakeholders involved in ATMP development about their perspective on the use of in vivo studies, the perceived hurdles and associated potential solutions regarding non-clinical development of ATMPs. In total, 17 stakeholders from 9 different countries were interviewed. A workshop was held with key stakeholders to further discuss major topics identified from the interviews. Conducting in vivo studies remains the status quo for ATMPs development. The hurdles identified included determining the amount of information required before clinical entry and effective use of limited human samples to understand a treatment or for clinical monitoring. A number of key points defined the need for future in vivo studies as well as improved application and implementation of New Approach Methodology (NAM)-based approach for products within a well-known modality or technology platform. These included data transparency, understanding of the added value of in vivo studies, and continuous advancement, evaluation, and qualification of NAMs. Based on the outcome of the discussions, a roadmap with practical steps towards a human-centric safety assessment of ATMPs was established.

Testing developmental toxicity in a second species: are the differences due to species or replication error?

Developmental toxicity studies for chemical and pharmaceutical safety are primarily performed in rats. Regulatory frameworks may require testing in a second, non-rodent species, for which the rabbit is usually chosen. This study shows that differences in NOAELs or LOAELs (N(L)OAELs) observed between rat and rabbit developmental toxicity studies performed according to OECD guidelines could just as well be caused by study replication errors, and not necessarily by differences in species sensitivity. This conclusion follows from an analysis of a database with rat and rabbit developmental toxicity studies for over 1000 industrial chemicals, pesticides, veterinary drugs and human pharmaceuticals, which included 143 compounds with multiple oral rat studies and 124 compounds with multiple oral rabbit studies. Our analysis confirms earlier findings that, on average over all compounds, rat and rabbit do not differ in sensitivity to developmental effects. There is substantial scatter in the correlation plots comparing rat and rabbit developmental N(L)OAELs, which is easily interpreted as species differences for individual compounds. However, for compounds tested twice in the same species, these N(L)OAELs may differ up to a factor of 25. Thus, potential interspecies differences in developmental N(L)OAEL will be overwhelmed by the reproducibility error, rendering the added value of a second species study questionable. As N(L)OAELs serve as point of departure (POD) for setting health-based guidance values in risk assessment, the large reproducibility error of N(L)OAELs should be taken into account by the introduction of an additional uncertainty factor. It is recommended to aim for reducing the reproducibility error by applying dose-response (BMD) analysis, optimize study designs and harmonize study protocols.

Analysis of exposure margins in developmental toxicity studies for detection of human teratogens.

The draft Step 2 ICH S5(R3) guideline includes an exposure-based endpoint as an option for selecting the high-dose in reproductive and developmental toxicity studies. To help determine an appropriate exposure margin for embryofetal developmental toxicity testing, a retrospective analysis was undertaken to determine what threshold would have been sufficient to detect hazards to embryofetal development in rats and rabbits for 18 known and 4 presumed human teratogens. The analysis showed that using a high dose that provided at least a 6-fold exposure margin in the developmental toxicity studies would have been sufficient to detect the teratogenic hazard with relevance for humans for all these therapeutics. With regards to human risk assessment practices for developmental toxicity, the analysis showed that, after excluding lenalidomide and pomalidomide data in rats, all available AUC margins at the NOAEL for the induction of malformations or embryofetal lethality were <4-fold of the exposure at the MRHD for all 22 therapeutics. These data support the proposed general approach of increased level of concern for human risk when exposure margins of the NOAEL to the MRHD are <10-fold, reduced concern when the exposure margins are 10- to 25-fold, and minimal concern when the exposure margin is > 25-fold.

Comparing rat and rabbit embryo-fetal developmental toxicity data for 379 pharmaceuticals: on systemic dose and developmental effects.

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.

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.

Comparison of gene expression regulation in mouse- and human embryonic stem cell assays during neural differentiation and in response to valproic acid exposure.

Embryonic stem cell tests (EST) are considered promising alternative assays for developmental toxicity testing. Classical mouse derived assays (mEST) are being replaced by human derived assays (hEST), in view of their relevance for human hazard assessment. We have compared mouse and human neural ESTn assays for neurodevelopmental toxicity as to regulation of gene expression during cell differentiation in both assays. Commonalities were observed in a range of neurodevelopmental genes and gene ontology (GO) terms. The mESTn showed a higher specificity in neurodevelopment than the hESTn, which may in part be caused by necessary differences in test protocols. Moreover, gene expression responses to the anticonvulsant and human teratogen valproic acid were compared. Both assays detected pharmacological and neurodevelopmental gene sets regulated by valproic acid. Common significant expression changes were observed in a subset of homologous neurodevelopmental genes. We suggest that these genes and related GO terms may provide good candidates for robust biomarkers of neurodevelopmental toxicity in hESTn.

Dynamic changes in energy metabolism upon embryonic stem cell differentiation support developmental toxicant identification.

Embryonic stem cells (ESC) are widely used to study embryonic development and to identify developmental toxicants. Particularly, the embryonic stem cell test (EST) is well known as in vitro model to identify developmental toxicants. Although it is clear that energy metabolism plays a crucial role in embryonic development, the modulation of energy metabolism in in vitro models, such as the EST, is not yet described. The present study is among the first studies that analyses whole genome expression data to specifically characterize metabolic changes upon ESC early differentiation. Our transcriptomic analyses showed activation of glycolysis, truncated activation of the tricarboxylic acid (TCA) cycle, activation of lipid synthesis, as well as activation of glutaminolysis during the early phase of ESC differentiation. Taken together, this energy metabolism profile points towards energy metabolism reprogramming in the provision of metabolites for biosynthesis of cellular constituents. Next, we defined a gene set that describes this energy metabolism profile. We showed that this gene set could be successfully applied in the EST to identify developmental toxicants known to modulate cellular biosynthesis (5-fluorouracil and methoxyacetic acid), while other developmental toxicants or the negative control did not modulate the expression of this gene set. Our description of dynamic changes in energy metabolism during early ESC differentiation, as well as specific identification of developmental toxicants modulating energy metabolism, is an important step forward in the definition of the applicability domain of the EST.

Valproic acid-induced gene expression responses in rat whole embryo culture and comparison across in vitro developmental and non-developmental models.

Transcriptomic evaluations may improve toxicity prediction of in vitro-based developmental models. In this study, transcriptomics was used to identify VPA-induced gene expression changes in rat whole embryo culture (WEC). Furthermore, VPA-induced responses were compared across in vitro-based developmental models, such as the cardiac and neural embryonic stem cells (ESTc and ESTn, respectively) and the zebrafish embryotoxicity model. VPA-induced gene regulation in WEC corresponded with observed morphological effects and previously suggested mechanisms of toxicity. Gene Ontology term-directed analysis showed conservation of VPA-induced gene expression changes across in vitro-based developmental models, with ESTc and ESTn exhibiting complementary responses. Furthermore, comparison of in vitro-based developmental and non-developmental models revealed that more generalized VPA-induced effects can be detected using non-developmental models whereas developmental models provide added value when assessing developmental-specific effects. These analyses can be used to optimize test batteries for the detection of developmental toxicants in vitro.

Complementary detection of embryotoxic properties of substances in the neural and cardiac embryonic stem cell tests.

In developmental toxicity testing, in vitro screening assays are highly needed to increase efficiency and to reduce animal use. A promising in vitro assay is the cardiac embryonic stem cell test (ESTc), in which the effect of developmental toxicants on cardiomyocyte differentiation is assessed. Recently, we developed a neural differentiation variant of the stem cell test (neural embryonic stem cell test [ESTn]). In both of these models, we have previously performed a series of transcriptomic studies to characterize gene expression changes (1) across time during normal differentiation and (2) in response to a series of developmental toxicants in the ESTn and ESTc. Here, using the cumulative of these studies, we compared gene expression profiles of ESTn and ESTc over time as well as model-specific changes induced by seven compounds, comprising six known in vivo developmental toxicants and one negative control. Time-related gene expression profiles showed similarities between the two EST systems. However, specific genes could be identified changing over time differently in each model related to the two different lineages of differentiation. Interestingly, compound-induced gene expression changes were generally model specific, especially for methylmercury and flusilazole, which were predicted better in ESTn and ESTc, respectively. Valproic acid-induced gene expression changes were most comparable out of the six developmental toxicants between the ESTn and ESTc. Direct transcriptomic comparisons between the ESTn and ESTc indicate that combined transcriptomic analyses support and complement each other. Therefore, a combined approach incorporating ESTc and ESTn may improve developmental toxicant detection over individual assays.

An optimized gene set for transcriptomics based neurodevelopmental toxicity prediction in the neural embryonic stem cell test.

The murine neural embryonic stem cell test (ESTn) is an in vitro model for neurodevelopmental toxicity testing. Recent studies have shown that application of transcriptomics analyses in the ESTn is useful for obtaining more accurate predictions as well as mechanistic insights. Gene expression responses due to stem cell neural differentiation versus toxicant exposure could be distinguished using the Principal Component Analysis based differentiation track algorithm. In this study, we performed a de novo analysis on combined raw data (10 compounds, 19 exposures) from three previous transcriptomics studies to identify an optimized gene set for neurodevelopmental toxicity prediction in the ESTn. By evaluating predictions of 200,000 randomly selected gene sets, we identified genes which significantly contributed to the prediction reliability. A set of 100 genes was obtained, predominantly involved in (neural) development. Further stringency restrictions resulted in a set of 29 genes that allowed for 84% prediction accuracy (area under the curve 94%). We anticipate these gene sets will contribute to further improve ESTn transcriptomics studies aimed at compound risk assessment.

Transcriptomic concentration-response evaluation of valproic acid, cyproconazole, and hexaconazole in the neural embryonic stem cell test (ESTn).

Alternative developmental toxicity assays are urgently needed to reduce animal use in regulatory developmental toxicology. We previously designed an in vitro murine neural embryonic stem cell test (ESTn) as a model for neurodevelopmental toxicity testing (Theunissen et al., 2010). Toxicogenomic approaches have been suggested for incorporation into the ESTn to further increase predictivity and to provide mechanistic insights. Therefore, in this study, using a transcriptomic approach, we investigated the concentration-dependent effects of three known (neuro) developmental toxicants, two triazoles, cyproconazole (CYP) and hexaconazole (HEX), and the anticonvulsant valproic acid (VPA). Compound effects on gene expression during neural differentiation and corresponding regulated gene ontology (GO) terms were identified after 24 h of exposure in relation to morphological changes on day 11 of culture. Concentration-dependent responses on individual gene expression and on biological processes were determined for each compound, providing information on mechanism and concentration-response characteristics. All compounds caused enrichment of the embryonic development process. CYP and VPA but not HEX significantly enriched the neuron development process. Furthermore, specific responses for triazole compounds and VPA were observed within the GO-term sterol metabolic process. The incorporation of transcriptomics in the ESTn was shown to enable detection of effects, which precede morphological changes and provide a more sensitive measure of concentration-dependent effects as compared with classical morphological assessments. Furthermore, mechanistic insight can be instrumental in the extrapolation of effects in the ESTn to human hazard assessment.

Innovative approaches in the embryonic stem cell test (EST).

The embryonic stem cell test (EST) is a high-throughput in vitro screening assay for developmental toxicity free of animal use. The EST uses the ability of murine embryonic stem cells to differentiate into the mesodermal cardiac lineage in combination with two cytotoxicity test systems. Validation of the EST showed that the test system is very promising as an alternative method to animal testing, however to optimize predictability and increase knowledge on the applicability domain of the EST, improvements to the method were proposed and studied. In this review we discuss the first definition of the EST followed by the innovative approaches which have been proposed to increase the predictivity of the EST, including implementation of molecular endpoints in the EST, such as omics technologies and the addition of alternative differentiation models to the testing paradigm, such as neural and osteoblast differentiation and the use of human stem cells. These efforts to improve the EST increase the value of embryonic stem cells used as in vitro systems to predict developmental toxicity.

Comparison of MeHg-induced toxicogenomic responses across in vivo and in vitro models used in developmental toxicology.

Toxicogenomic evaluations may improve toxicity prediction of in vitro-based developmental models, such as whole embryo culture (WEC) and embryonic stem cells (ESC), by providing a robust mechanistic marker which can be linked with responses associated with developmental toxicity in vivo. While promising in theory, toxicogenomic comparisons between in vivo and in vitro models are complex due to inherent differences in model characteristics and experimental design. Determining factors which influence these global comparisons are critical in the identification of reliable mechanistic-based markers of developmental toxicity. In this study, we compared available toxicogenomic data assessing the impact of the known teratogen, methylmercury (MeHg) across a diverse set of in vitro and in vivo models to investigate the impact of experimental variables (i.e. model, dose, time) on our comparative assessments. We evaluated common and unique aspects at both the functional (Gene Ontology) and gene level of MeHg-induced response. At the functional level, we observed stronger similarity in MeHg-response between mouse embryos exposed in utero (2 studies), ESC, and WEC as compared to liver, brain and mouse embryonic fibroblast MeHg studies. These findings were strongly correlated to the presence of a MeHg-induced developmentally related gene signature. In addition, we identified specific MeHg-induced gene expression alterations associated with developmental signaling and heart development across WEC, ESC and in vivo systems. However, the significance of overlap between studies was highly dependent on traditional experimental variables (i.e. dose, time). In summary, we identify promising examples of unique gene expression responses which show in vitro-in vivo similarities supporting the relevance of in vitro developmental models for predicting in vivo developmental toxicity.

Time-response evaluation by transcriptomics of methylmercury effects on neural differentiation of murine embryonic stem cells.

Current globally harmonized Organisation for Economic Co-operation and Development (OECD) animal test guidelines for developmental toxicity require high numbers of experimental animals. To reduce animal use in this field, alternative developmental toxicity assays are highly desirable. We previously developed a dynamic in vitro model for screening effects of possible neurodevelopmental toxicants, using neural cell differentiation of pluripotent murine embryonic stem cells. To further mechanistically characterize the mouse neural embryonic stem cell test (ESTn) and to improve detection of possible neurodevelopmental toxicants, gene expression patterns were studied describing neural cell differentiation over time, as well as the impact on gene expression of exposure to the well-known neurotoxicant methylmercury (MeHg). A transcriptomics study was performed to examine whole-genome expression changes during the first 7 days of the cell differentiation protocol. Specific gene clusters were identified and enrichment analysis of Gene Ontology (GO) terms and gene sets derived from literature was performed using DAVID and T-profiler. Over time, a decrease of blastocyst and trophectoderm GO terms was observed, which included well-characterized pluripotency genes. Furthermore, an increase in the range of neural development-related GO terms, such as neuron differentiation and the wnt pathway, was observed. Analysis of gene expression using principle component analysis showed a time-dependent track in untreated cells, describing the process of neural differentiation. Furthermore, MeHg was shown to induce deviation from the predefined differentiation track. The compound inhibited general development GO terms and induced neural GO terms over time. This system appears promising for studying compound effects on neural differentiation in a mechanistic approach.