Developmental And Reproductive Toxicity Research Articles

Retinoid signaling in skeletal development: Scoping the system for predictive toxicology

All-trans retinoic acid (ATRA), the biologically active form of vitamin A, is instrumental in regulating the patterning and specification of the vertebrate embryo. Various animal models demonstrate adverse developmental phenotypes following experimental retinoid depletion or excess during pregnancy. Windows of vulnerability for altered skeletal patterning coincide with early specification of the body plan (gastrulation) and regional specification of precursor cell populations forming the facial skeleton (cranial neural crest), vertebral column (somites), and limbs (lateral plate mesoderm) during organogenesis. A common theme in physiological roles of ATRA signaling is mutual antagonism with FGF signaling. Consequences of genetic errors or environmental disruption of retinoid signaling include stage- and region-specific homeotic transformations to severe deficiencies for various skeletal elements. This review derives from an annex in Detailed Review Paper (DRP) of the OECD Test Guidelines Programme (Project 4.97) to support recommendations regarding assay development for the retinoid system and the use of resulting data in a regulatory context for developmental and reproductive toxicity (DART) testing.

Setting occupational exposure limits for antimicrobial agents: A case study based on a quaternary ammonium compound-based disinfectant

Antimicrobial agents have become an essential tool in controlling the transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and guidelines on their use have been issued by various public health agencies. Through its Emerging Viral Pathogen Guidance for Antimicrobial Pesticides, the US Environmental Protection Agency has approved numerous surface disinfectant products for use against SARS-CoV-2. Despite their widespread use and range of associated health hazards, the majority of active ingredients in antimicrobial products, such as surface disinfectants, lack established occupational exposure limits (OELs) to assist occupational health professionals in characterizing risks from exposures to these chemicals. Based on established approaches from various organizations, a framework for deriving OELs specific to antimicrobial agents was developed that relies on a weight-of-evidence evaluation of the available data. This framework involves (1) a screening-level toxicological assessment based on a review of the existing literature and recommendations, (2) identification of the critical adverse effect(s) and dose–response relationship(s), (3) identification of alternative health-based exposure limits (HBELs), (4) derivation of potential OELs based on identified points of departure and uncertainty factors and/or modification of existing alternative HBELs, and (5) selection of an appropriate OEL. To demonstrate the use of this framework, a case study is described for selection of an OEL for a disinfectant product containing quaternary ammonium compounds (quats). Three potential OELs were derived for this product based on irritation toxicity data, developmental and reproductive toxicity (DART) data, and modification of an existing HBEL. The final selected OEL for the quats-containing product was 0.1 mg/m3, derived from modification of an existing HBEL. This value represented the lowest resulting value of the three approaches, and thus, was considered protective of irritation and potential DART.

Incorporation of in vitro techniques for botanicals dietary supplement safety assessment – Towards evaluation of developmental and reproductive toxicity (DART)

As complex mixtures, botanicals present unique challenges when assessing safe use, particularly when endpoint gaps exist that cannot be fully resolved by existing toxicological literature. Here we explore in vitro gene expression as well receptor binding and enzyme activity as alternative assays to inform on developmental and reproductive toxicity (DART) relevant modes of action, since DART data gaps are common for botanicals. Specifically, botanicals suspected to have DART effects, in addition to those with a significant history of use, were tested in these assays. Gene expression changes in a number of different cell types were analysed using the connectivity mapping approach (CMap) to identify modes of action through a functional read across approach. Taken together with ligand affinity data obtained using a set of molecular targets customised towards known DART relevant modes of action, it was possible to inform DART risk using functional analogues, potency comparisons and a margin of internal exposure approach.

Value and limitation of structure-based profilers to characterize developmental and reproductive toxicity potential

The uncertainty regarding the safety of chemicals leaching from food packaging triggers attention. In silico models provide solutions for screening of these chemicals, since many are toxicologically uncharacterized. For hazard assessment, information on developmental and reproductive toxicity (DART) is needed. The possibility to apply in silico toxicology to identify and quantify DART alerts was investigated. Open-source models and profilers were applied to 195 packaging chemicals and analogues. An approach based on DART and estrogen receptor (ER) binding profilers and molecular docking was able to identify all except for one chemical with documented DART properties. Twenty percent of the chemicals in the database known to be negative in experimental studies were classified as positive. The scheme was then applied to 121 untested chemicals. Alerts were identified for sixteen of them, five being packaging substances, the others structural analogues. Read-across was then developed to translate alerts into quantitative toxicological values. They can be used to calculate margins of exposure (MoE), the size of which reflects safety concern. The application of this approach appears valuable for hazard characterization of toxicologically untested packaging migrants. It is an alternative to the use of default uncertainty factor (UF) applied to animal chronic toxicity value to handle absence of DART data in hazard characterization.

Exposure assessments in reproductive and developmental toxicity testing: An IQ-DruSafe industry survey on current practices and experiences in support of exposure-based high dose selection

The draft ICH S5(R3) guideline includes an exposure-based endpoint as an option for selecting the high dose in developmental and reproductive toxicity (DART) studies. In 2016, IQ DruSafe conducted an anonymous survey to identify industry practices and experiences related to pharmacokinetic assessments in DART studies in order to facilitate a pragmatic data-driven approach to development of an acceptable multiple of the clinical exposure to be proposed for dose selection in the guideline. Questions in the survey were designed to explore pharmacokinetic differences in pregnant versus non-pregnant animals, and to assess exposure levels attained in the absence of maternal toxicity as well as DART outcomes in animal studies associated with those exposures. Small molecule and therapeutic proteins were analyzed separately. The key findings for small molecules were: a) differences in exposures between pregnant and non-pregnant animals were generally ≤3-fold, b) Cmax or AUC exposures ≥25-fold the clinical exposure were achieved in the absence of maternal toxicity for 31% and 23% of rat and rabbit developmental toxicity studies, respectively, and c) only 3.3% (5/153) and 1.6% (2/128) of the developmental toxicity studies were positive for malformations or embryofetal lethality in rats and rabbits, respectively, that were not observed until exposure margins were ≥25-fold.

Soy-deficient diet induces renal lesions in juvenile rats

Certified LabDiet® 5K96 Advanced Protocol™ Verified Casein Diet 10 IF (5K96) is a commercial diet low in soy isoflavones developed for use in developmental and reproductive toxicity (DART) studies, especially those designed to detect endocrine disruptors. The objective of this study was to determine the incidences and severities of 5K96-associated renal lesions in control F0 and F1 cohorts of rats fed the 5K96 diet. Kidneys from control animals of four DART studies involving Sprague-Dawley rats fed the 5K96 diet, were evaluated microscopically. Mineralization and basophilic tubules were present in high incidence/severity in males and females compared to historical controls fed conventional diets. F1 cohorts were affected to a far greater degree than F0 cohorts, and females were affected more than males. Consideration of target tissue and mode of action should be given before automatically incorporating the 5K96 diet into DART study designs, and caution should be exercised when identifying and interpreting renal toxicity in the F1 cohorts of such studies.

New animal-free concepts and test methods for developmental toxicity and peripheral neurotoxicity.

The complex toxicological fields of repeat dose organ toxicity (RDT) and developmental and reproductive toxicity (DART) still require new concepts and approaches to achieve a fully animal-free safety assessment of chemicals. One novel approach is the generation of relevant human cell types from pluripotent stem cells, and the use of such cells for the establishment of phenotypic test methods. Due to their broad endpoints, such tests capture multiple types of toxicants, i.e. they are a readout for the activation of many adverse outcome pathways (AOPs). The 2016 Lush Science Prize was awarded for the development of one such assay, the PeriTox test, which uses human peripheral neurons generated from stem cells. The assay endpoints measure various cell functions, and these give information on the potential neurotoxicity and developmental neurotoxicity hazard of test compounds. The PeriTox test method has a high predictivity and sensitivity for peripheral neurotoxicants, and thus addresses the inherent challenges in pesticide testing and drug development. Data from the test can be obtained quickly and at a relatively high-throughput, and thus, the assay has the potential to replace animal-based safety assessment during early product development or for screening potential environmental toxicants.

An enhanced C. elegans based platform for toxicity assessment

There is a well-defined regulatory framework governing the approval of chemicals for use as pharmaceuticals or release into the environment. Toxicity assessment is thus a major hurdle in the compound discovery pipeline, currently involving large scale animal testing. The search for alternative testing platforms is therefore an important priority. We have developed a convenient, low cost assay utilising the nematode Caenorhabditis elegans, to rapidly assess both acute toxicity and developmental and reproductive toxicity (DART). However the worm is protected by a robust cuticle that forms a barrier to chemical uptake. We assessed mutants with altered cuticle properties to identify sensitized strains optimized for toxicity assays. Evaluating the trade-off between increased permeability and reduced fitness identifies bus-5(br19) as the most suitable strain for chemical exposure. We demonstrate the applicability of this assay for a range of chemicals with differing properties, including a modified exposure protocol for volatile or less soluble compounds. This work enhances the effectiveness of C. elegans for convenient toxicity assessment, which could contribute to a reduction in the use of vertebrates particularly at the crucial early stages of product development. Strains identified in this work will also enhance the sensitivity of C. elegans based drug discovery platforms.

Reproductive and developmental toxicology studies with gantenerumab in PS2APP transgenic mice

Gantenerumab is intended for the treatment of Alzheimer’s disease. It is a fully human recombinant monoclonal IgG1 which binds aggregated forms of amyloid beta such as Aβ oligomers, fibrils and neuritic amyloid plaques. A full package of developmental and reproductive toxicity (DART) studies was performed in the PS2APP double-transgenic mouse model of Alzheimer’s disease. A combined fertility and embryo fetal development study and a pre-and post-natal development study were performed. The PS2APP mouse model allowed a more complete DART evaluation than would have been possible in conventional species or strains which do not express the target antigen of gantenerumab. No developmental or reproductive hazards were identified.

Influence of Study Design on Developmental and Reproductive Toxicology Study Outcomes.

Regulatory studies of developmental and reproductive toxicity (DART) studies have remained largely unchanged for decades, with exposures occurring at various phases of the reproductive cycle and toxicity evaluations at different ages/times depending on the study purpose. The National Toxicology Program has conducted studies examining the power to detect adverse effects where there is a prenatal exposure, but evaluations occur postnatally. In these studies, examination is required of only 1 male and female pup from each litter beyond weaning. This provides poor resolving power to detect rare events (e.g., reproductive tract malformations). If an adverse effect is detected, there is little confidence in the shape of the dose-response curve (and the Benchmark Dose or No Observed Adverse Effect Level [NOAEL]). We have developed a new protocol to evaluate DART, the modified one generation study, with exposure commencing with pregnant animals and retention of 4 males and females from each litter beyond weaning to improve statistical power. These animals can be allocated to specific cohorts that examine subchronic toxicity, teratology, littering, and neurobehavioral toxicity in the same study. This approach also results in a reduction in animal numbers used, compared with individual stand-alone studies, and offers increased numbers of end points evaluated compared with recent Organization for Economic Cooperation and Development proposals.

Special endpoint and product specific considerations in pharmaceutical acceptable daily exposure derivation

Recently, a guideline has been published by the European Medicines Agency (EMA) on setting safe limits, permitted daily exposures (PDE) [also called acceptable daily exposures (ADE)], for medicines manufactured in multi-product facilities. The ADE provides a safe exposure limit for inadvertent exposure of a drug due to cross-contamination in manufacturing. The ADE determination encompasses a standard risk assessment, requiring an understanding of the toxicological and pharmacological effects, the mechanism of action, drug compound class, and the dose-response as well as the pharmacokinetic properties of the compound. While the ADE concept has broad application in pharmaceutical safety there are also nuances and specific challenges associated with some toxicological endpoints or drug product categories. In this manuscript we discuss considerations for setting ADEs when the following specific adverse health endpoints may constitute the critical effect: genotoxicity, developmental and reproductive toxicity (DART), and immune system modulation (immunostimulation or immunosuppression), and for specific drug classes, including antibody drug conjugates (ADCs), emerging medicinal therapeutic compounds, and compounds with limited datasets. These are challenging toxicological scenarios that require a careful evaluation of all of the available information in order to establish a health-based safe level.

Threshold of toxicological concern (TTC) for developmental and reproductive toxicity of anticancer compounds

Pharmaceutical companies develop specialized therapies to treat late stage cancer. In order to accelerate life-saving treatments and reduce animal testing, compounds to treat life-threatening malignancies are allowed modified requirements for preclinical toxicology testing. Limited data packages in early drug development can present product quality challenges at multi-product manufacturing facilities. The present analysis established an endpoint-specific threshold of toxicological concern (TTC) for developmental and reproductive toxicity (DART) for anticancer compounds. A comprehensive database was created consisting of over 300 no-observed adverse effect levels (NOAELs) for DART of 108 anticancer compounds. The 5th percentile NOAEL for developmental and reproductive toxicity was 0.005mg/kg/day (300μg/day), resulting in a human exposure threshold of 3μg/day assuming standard uncertainty factors and a 60kg human bodyweight. The analysis shows this threshold is protective for developmental and reproductive toxicity of highly potent groups of anticancer compounds. There were similar TTC values calculated for direct-acting and indirect-acting anticancer compounds.

Framework for Identifying Chemicals with Structural Features Associated with the Potential to Act as Developmental or Reproductive Toxicants

Developmental and reproductive toxicity (DART) end points are important hazard end points that need to be addressed in the risk assessment of chemicals to determine whether or not they are the critical effects in the overall risk assessment. These hazard end points are difficult to predict using current in silico tools because of the diversity of mechanisms of action that elicit DART effects and the potential for narrow windows of vulnerability. DART end points have been projected to consume the majority of animals used for compliance with REACH; thus, additional nonanimal predictive tools are urgently needed. This article presents an empirically based decision tree for determining whether or not a chemical has receptor-binding properties and structural features that are consistent with chemical structures known to have toxicity for DART end points. The decision tree is based on a detailed review of 716 chemicals (664 positive, 16 negative, and 36 with insufficient data) that have DART end-point data and are grouped into defined receptor binding and chemical domains. When tested against a group of chemicals not included in the training set, the decision tree is shown to identify a high percentage of chemicals with known DART effects. It is proposed that this decision tree could be used both as a component of a screening system to identify chemicals of potential concern and as a component of weight-of-evidence decisions based on structure-activity relationships (SAR) to fill data gaps without generating additional test data. In addition, the chemical groupings generated could be used as a starting point for the development of hypotheses for in vitro testing to elucidate mode of action and ultimately in the development of refined SAR principles for DART that incorporate mode of action (adverse outcome pathways).

S5-3: Bottlenecks for the implementation of alternative methods in regulatory reproductive and developmental toxicity testing

Since adoption of the first globally implemented guidelines for developmental and reproductive toxicity (DART) testing for pharmaceuticals, industrial chemicals and agrochemicals, many years passed without major updates. However in recent years, significant changes in these guidelines have been made or are being implemented. These changes have been guided by the ethical drive to reduce, refine and replace (3R) animal testing, as well as the addition of endocrine disruptor relevant endpoints. Recent applied improvements have focused on reduction and refinement. Ongoing scientific and technical innovations will provide the means for replacement of animal testing in the future and will improve predictivity in humans. The aim of this review is to provide an overview of ongoing global DART endeavors in respect to the 3Rs, with an outlook towards future advances in DART testing aspiring to reduce animal testing to a minimum and the supreme ambition towards animal-free hazard and risk assessment.

A critical evaluation of developmental and reproductive toxicology in nonhuman primates

The nonhuman primates (NHPs) are used in many areas of biomedical research where their similarities to humans make them exclusively valuable animal models. The use of NHPs in pre-clinical testing is expected to increase due to the increase in the development of biological compounds for therapeutic uses. The regulatory agencies around the world including Food and Drug Administration (FDA) generally requires developmental and reproductive toxicity (DART) testing of all new drugs to be used by women of childbearing age or men of reproductive potential. NHPs are most frequently used for DART testing when commonly used rodents and//or rabbits are not pharmacologically relevant species. Animal studies are unique in that assessment of reproduction and development as DART studies are not performed in controlled clinical trials; therefore, pre-clinical safety assessment forms the basis for risk assessment for marketed drug products. This paper provides a critical evaluation of developmental and reproductive toxicity studies in NHPs. The manuscript will focus on species selection, limitation of International Conference for Harmonization stages (A-F) using NHPs as a test system, study designs, logistical//technical challenges, and strength, and limitations. It will also pinpoint confounding factors inherent to the test system that may complicate the interpretation of the NHP DART data.

Case studies to test: A framework for using structural, reactivity, metabolic and physicochemical similarity to evaluate the suitability of analogs for SAR-based toxicological assessments

A process for evaluating analogs for use in SAR (Structure–Activity Relationship) assessments was previously published ( Wu et al. 2010). Subsequently, this process has been updated to include a decision tree for estrogen binding (from US EPA) and flags for developmental and reproductive toxicity (DART). This paper presents the results of blinded case studies designed to test this updated framework. The results of these case studies support the conclusion that the process outlined by Wu et al. (2010) can be successfully applied to develop surrogate values for risk assessment. The read across results generated by the process were shown to be protective when compared to the actual toxicity data. Successful application of the approach requires significant expertise as well as discipline to not overstep the boundaries of the defined analogs and the rating system. The end result of this rigor can be the inability to read across all endpoints for all chemicals resulting in data gaps that cannot be filled using read across, however, this reflects the current state of the science and is preferable to making non-protective decisions. Future work will be targeted towards expanding read across capabilities. Two examples of a broader category approach are also shown.

Ethyl t‐butyl ether: review of reproductive and developmental toxicity

Ethyl t-butyl ether (ETBE) is a motor fuel oxygenate used in reformulated gasoline. Knowledge of developmental and reproductive toxicity potential of ETBE is critical for making informed decisions about acceptance and regulations. This review discusses toxicology studies providing information about effects on reproduction and the conceptus. Seven GLP-compliant studies following widely accepted protocols have focused specifically on developmental and reproductive toxicity (DART) in rats and rabbits exposed to ETBE by gavage with doses up to 1,000 mg/kg body weight/day, the limit specified in standardized test guidelines. Other repeat-dose general toxicology studies have administered ETBE to rodents for up to 180 days, and included reproductive organ weights, histology, or other indications of reproductive system structure or function. DART potential of the main ETBE metabolite t-butyl alcohol and class-related MTBE has also been studied. More GLP-compliant studies exist for evaluating ETBE using well-established, currently recommended protocols than are available for many other chemicals used today. The database for determining ETBE DART potential is adequate, although not all study details are currently easily accessible for peer-review. ETBE does not appear to be selectively toxic to reproduction or embryofetal development in the absence of other manifestations of general toxicity. Studies using recommended methods for sample preservation and analysis have shown no targeted effect on the reproductive system. No embryofetal effects were observed in rabbits. Early postnatal rat pup deaths show no clear dose-response and have largely been attributed to total litter losses with accompanying evidence of maternal neglect or frank maternal morbidity.

Considerations in assessing the developmental and reproductive toxicity potential of biopharmaceuticals

This report discusses the principles of developmental and reproductive toxicity (DART) testing for biopharmaceuticals. Biopharmaceuticals are large-molecular-weight proteins or peptides produced by modern biotechnology techniques incorporating genetic engineering and hybridoma technologies. The principles of DART testing for biopharmaceuticals are similar to those for small-molecule pharmaceuticals and in general follow the regulatory guidance outlined in International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) document S5(R2). However, because many biopharmaceuticals are species-specific, alternate approaches may be needed to evaluate DART potential as outlined in ICH S6. For molecules that show species-specific cross-reactivity restricted to non-human primates (NHP), some aspects of DART may require NHP testing. For biopharmaceuticals that are uniquely specific and only active on intended human targets or human and chimpanzee targets, surrogate molecules that cross-react with the more traditional rodent species may need to be developed and used for DART testing. Alternatively, genetically modified transgenic animals may also need to be considered. Surrogate molecules and transgenic animals may also be considered for DART testing even if the biopharmaceutical is active in NHPs in order to reduce the use of NHPs. Because of the unique properties of biopharmaceuticals, a case-by-case approach is needed for DART and general toxicity evaluation, which requires consideration of specific product attributes including biochemical and biophysical characteristics, pharmacological activity, and intended clinical indication.

Nonclinical Aspects of Biopharmaceutical Development: Discussion of Case Studies at a PhRMA-FDA Workshop

Robust assessments of the nonclinical safety profile of biopharmaceuticals are best developed on a scientifically justified, case-by-case basis, with consideration of the therapeutic molecule, molecular target, and differences/similarities between nonclinical species and humans (ICH S6). Significant experience has been gained in the 10 years ensuing since publication of the ICH S6 guidance. In a PhRMA-FDA-sponsored workshop, "Nonclinical Aspects of Biopharmaceutical Development," industry and US regulatory representatives engaged in exploration of current scientific and regulatory issues relating to the nonclinical development of biopharmaceuticals in order to share scientific learning and experience and to work towards establishing consistency in application of general principles and approaches. The proceedings and discussions of this workshop confirm general alignment of strategy and tactics in development of biopharmaceuticals with regard to such areas as species selection, selection of high doses in toxicology studies, selection of clinical doses, the conduct of developmental and reproductive toxicity (DART) studies, and assessment of carcinogenic potential. However, several important aspects, including, for example, appropriate use of homologues, nonhuman primates, and/or in vitro models in the assessment of risk for potential developmental and carcinogenic effects, were identified as requiring further scientific exploration and discussion.

Application of the Tukey Trend Test Procedure to Assess Developmental and Reproductive Toxicity: I. Measurement Data

Application of the Tukey Trend Test Procedure to Assess Developmental and Reproductive Toxicity. I. Measurement Data. Antonello, J. M., Clark, R. L., and Heyse, J. F. (1993). Fundam. Appl. Toxicol. 21, 52-58.Developmental and reproductive (DAR) toxicity studies typically include a series of increasing doses of a compound and zero dose control. Given this framework, Tukey et al. (Biometrics, 41, 295-301, 1985) proposed a procedure (referred to as either the Tukey trend or TCH test procedure) for detecting a nonzero trend in response to increasing doses of the test compound. The procedure considers three candidate dosage scalings to ensure high power against relatively common dose-response patterns and appreciable power against most reasonable patterns. For toxicologic effects with near monotonic dose-response patterns, simulation studies have shown the TCH test to be overall more powerful than pairwise comparison procedures. The TCH test can be applied sequentially, eliminating the highest dose each time a statistically significant trend is observed, until a no-statistical-significance-of trend dose is reached. This is the highest dose through which there is no statistically trustworthy evidence of the compound's impact on the response. Since DAR toxicity usually exhibits a progressive (monotonic) dose-response, we advocate routine use of Tukey's trend test for the evaluation of treatment effects in these studies. In this article, we discuss the procedure in detail and apply it to fetal body weight, a continuous measurement variable, from a developmental toxicity study.