Background This report addresses the reliability of results from rat Embryo-Fetal Developmental Toxicity (EFDT) studies. Recent literature discusses the roles of reproducibility, replicability, and other influences on scientific reliability. Reproducibility is a re-analysis of the original data, while replicability addresses the same question with a separate study of some type. Concordance of rat and rabbit studies has been addressed previously, but replication of single-species EFDT studies was not found in the literature. A modest modification of the rat study is therefore proposed to assess replicability and possibly enhance reliability. Methods Regulatory guidelines were consulted and relevant literature was identified through online searches. Results Each replicate EFDT (r-EFDT) study in rats would consist of half the mated females of the definitive study. Studies would start at the same or different times in one testing facility. Separate shipments of animals (non-littermates) are required. All other procedures would be protocol-driven. The micro- and macro-environments of the animals would be held as constant as possible. Justification, design options, and interpretation methods are discussed. Conclusion Besides adding reliability, other benefits include reduced animal usage, and potentially reduced cost and time to final reports. By reducing the need for repeated studies due to questionable results, this modified study is viewed as a more efficient use of costly resources. The r-EFDT study design could easily be adapted to assess replicability of rabbit EFDT and some general toxicity studies. Future replicate studies are needed to critically evaluate replicability and the overall impact on study reliability.

The use of virtual control groups (VCGs) in nonclinical toxicology studies was first proposed in 2020 with the main purpose of reducing animal use while integrating historical control data (HCD) to enhance study interpretation. The use of VCGs has gained increasing attention as evidenced by an increasing number of publications that highlight implementation challenges. Laboratories that conduct harmonized studies with standardized procedures, consistent environmental conditions, and validated electronic databases are well-suited to implement VCGs in future nonclinical safety studies. We suggest that individual laboratories conducting rodent and rabbit developmental and reproductive toxicity studies should begin planning for VCG implementation. If possible, a harmonized approach to VCG implementation by multiple laboratories will lend credence to regulatory approval. We apply the six-step VCG implementation framework from Palazzi et al. to the routine GLP studies covered by international guidelines, which emphasize validation through retrospective and prospective trials. We discuss the risks and challenges to VCG implementation that have been previously presented. To address some of these concerns, a hybrid approach is proposed that combines a small concurrent control group (CCG) with multiple virtual control (VC) animals from the same test facility. The inclusion of a CCG addresses the need to monitor for disease and environmental changes and prevent depletion of HCD. Two approaches to the selection of VC animals are discussed. Given that developmental and reproductive toxicity studies use the most animals in nonclinical safety studies, we support the timely implementation of VCGs to significantly reduce these animal numbers.

INTRODUCTION:A key aspect of scientific reliability includes replicability, that is, obtaining consistent results when an experiment is repeated. In embryo-fetal developmental toxicity (EFDT) studies, replicability can be assessed using in vitro models, targeted in vivo studies, and/or the second species study. This work assesses the replicability of whole-animal studies using historic rat data. METHODS:Data for two endpoints from five full studies were downloaded from the National Toxicology Program (NTP) website. Each full group was divided into two replicate sets (based on odd/even and top/bottom animal order) to evaluate within-study replicability. Analyses included summary statistics, scatter plots, a modified Levene's test for homogeneity of variances, and Cohen's d to assess effect sizes. RESULTS:Replicate means deviated from the original study by only 0.4%-3.7% and differed by ≤ 7% between replicates (with differences < 5% in 87% of groups). Coefficients of variation (CV%) were generally consistent across subgroups, with few above 10%. Variance testing revealed significant differences in two of the five studies, and one study exhibited opposite fetal weight effects in the odd/even subgroup only. Evaluations of adjusted maternal weight gain were comparable across subgroups. CONCLUSIONS:The observed 5%-7% differences between these idealized replicates may represent the lower bound for acceptable variability when merging replicate data sets. This work lays the groundwork for more robust evaluations of replicability in EFDT studies and may inform future regulatory guidance.

BackgroundMeasurement of rat anogenital distance (AGD) dates to at least 1912. Increased interest in endocrine disrupting chemicals and the use of AGD as a biomarker for fetal androgen effects have increased the number of studies with this endpoint in recent decades. A literature review revealed different landmarks, methods of measurement, and methods to adjust for body weight differences. AGD is often reported to hundredths of millimeters and as such, deserves precision in all these aspects. This paper presents recommendations for the measurement and analysis of rodent AGD.MethodsLiterature and regulatory guidance documents that mentioned or measured rodent AGD were reviewed. Four adjustment methods were evaluated using available online data from three rat studies each with two generations of offspring.ResultsTabulation of studies reveals that species/stocks and time of data collection, but more importantly anatomical landmarks and methods of measurement have produced a variety of results which are difficult to compare. Not all studies have adjusted for test article effects on body weight (and thus size). The four adjustment methods were fairly comparable.ConclusionRecommendations are as follows. A microscopic method should be used to measure AGD of late rodent fetuses and early postnatal pups. The caudal edge of the genital tubercle and the cranial edge of the anus are clear and identifiable landmarks. The simplest adjustment is to divide individual AGDs by the cube root of animals' body weight. These recommendations will help ensure data consistency and accuracy, and facilitate meaningful comparisons across laboratories and chemical classes.

Birth Defects ResearchVolume 116, Issue 3 e2326 CORRESPONDENCE Comments on "Maternal–fetal safety evaluation of an aqueous extract of Casearia sylvestris [AECS] leaves in rats" (Nagaoka et al., 2023 [DOI: 10.1002/bdr2.2257]) L. David Wise, Corresponding Author L. David Wise [email protected] orcid.org/0000-0002-2280-0725 Independent Teratologist, Philadelphia, Pennsylvania, USA Correspondence L. David Wise, Independent Teratologist, Philadelphia, PA, USA. Email: [email protected]Search for more papers by this authorJohn M. DeSesso, John M. DeSesso orcid.org/0000-0002-3134-4207 Exponent, Inc., Alexandria, Virginia, USASearch for more papers by this author L. David Wise, Corresponding Author L. David Wise [email protected] orcid.org/0000-0002-2280-0725 Independent Teratologist, Philadelphia, Pennsylvania, USA Correspondence L. David Wise, Independent Teratologist, Philadelphia, PA, USA. Email: [email protected]Search for more papers by this authorJohn M. DeSesso, John M. DeSesso orcid.org/0000-0002-3134-4207 Exponent, Inc., Alexandria, Virginia, USASearch for more papers by this author First published: 23 March 2024 https://doi.org/10.1002/bdr2.2326Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Open Research DATA AVAILABILITY STATEMENT Data sharing is not applicable to this article as no new data were created or analyzed in this study. REFERENCES Charles River. (n.d.). Historical control data [HCD]. Reproductive toxicology data, rats (e.g., Netherlands, Pennsylvania, Edinburgh, Ohio). https://www.criver.com/products-services/safety-assessment/toxicology-services/developmental-and-reproductive-toxicology-dart/historical-control-data?region=3601 Google Scholar Clark, R. L., Antonello, J., Grossman, S., Wise, L. D., Anderson, C., Bagdon, W. J., Prahalada, S., MacDonald, J. S., & Robertson, R. T. (1990). External genitalia abnormalities in male rats exposed in utero to finasteride, a 5α-reductase inhibitor. Teratology, 42, 91–100. https://doi.org/10.1002/tera.1420420111 10.1002/tera.1420420111 CASPubMedWeb of Science®Google Scholar DeSesso, J. M., & Scialli, A. R. (2018). Bone development in laboratory mammals used in developmental toxicity studies. Birth Defects Research, 110, 1157–1187. https://doi.org/10.1002/bdr2.1350 10.1002/bdr2.1350 CASPubMedWeb of Science®Google Scholar Gallavan, R. H., Holson, J. F., Stump, D. G., Knapp, J. F., & Reynolds, V. L. (1999). Interpreting the toxicologic significance of alterations in anogenital distance: Potential for confounding effects of progeny body weights. Reproductive Toxicology, 13(5), 383–390. https://doi.org/10.1016/s0890-6238(99)00036-2 10.1016/S0890-6238(99)00036-2 CASPubMedWeb of Science®Google Scholar Organisation for Economic Cooperation and Development. (2018). Test no. 414: Prenatal developmental toxicity study. In OECD guidelines for the testing of chemicals, section 4. OECD Publishing. https://doi.org/10.1787/9789264070820-en Google Scholar Reagan-Shaw, S., Nihal, M., & Ahmad, N. (2008). Dose translation from animal to human studies revisited. The FASEB Journal, 22, 659–661. https://doi.org/10.1096/fj.07-9574LSF 10.1096/fj.07-9574LSF CASPubMedWeb of Science®Google Scholar Wise, L. D., Buschmann, J., Feuston, M. H., Fisher, J. E., Hew, K. W., Hoberman, A. M., Lerman, S. A., Ooshima, Y., & Stump, D. G. (2009). Embryo-fetal developmental toxicity study design for pharmaceuticals. Birth Defects Research. Part B: Developmental and Reproductive Toxicology, 86(6), 418–428. https://doi.org/10.1002/bdrb.20214 10.1002/bdrb.20214 CASPubMedWeb of Science®Google Scholar Wise, L. D., Vetter, C. M., Anderson, C. A., Antonello, J. M., & Clark, R. L. (1991). Reversible effects of triamcinolone and lack of effects with aspirin or L-656,224 on external genitalia of male Sprague–Dawley rats exposed in utero. Teratology, 44(5), 507–520. https://doi.org/10.1002/tera.1420440505 10.1002/tera.1420440505 CASPubMedWeb of Science®Google Scholar Volume116, Issue3March 2024e2326 ReferencesRelatedInformation

The authors report no conflict of interest. Data S1. Supporting information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

GENERAL COMMENTARY article Front. Toxicol., 04 December 2023Sec. Developmental and Reproductive Toxicology Volume 5 - 2023 | https://doi.org/10.3389/ftox.2023.1293873

BackgroundA new derived (i.e., calculated) endpoint of developmental toxicology has appeared in a very few studies since 1990. This endpoint is adjusted mean live fetal weight per litter or adjusted fetal weight. Given our lack of familiarity with the endpoint, we evaluated the basis, prevalence, methods, and usefulness in embryo-fetal developmental toxicity (EFDT) studies in rats.MethodsLiterature searches were performed with key terms using PubMed and Google Scholar. Major textbooks were consulted but lack of any mention of the endpoint. Unpublished EFDT data, which are readily available online, were utilized to test adjustment methods.ResultsPertinent information on factors that influence fetal weight goes back a century. Four papers utilizing rats were found in which fetal weights were adjusted using either statistical or formula-based methods to adjust fetal weights. Only one study showed a clear benefit to the endpoint when there was a marked decrease in live litter size; this pointed to situations in which the new endpoint might be useful. The lone formula-based adjustment method was found to be lacking adequate testing and justifications. A new experimental alternative formula-based adjustment is shown to produce results very similar to statistical methods.ConclusionsFrom this assessment, we recommend that adjusted fetal weight should not be a routine endpoint at this time. However, there are likely cases where this derived endpoint could aid interpretation. We encourage other investigators to examine previous EFDT study data to establish guidance on the use of adjusted mean live fetal weights.

There are incorrect statements in the Discussion. The study Results are inappropriately presented and interpreted. There are several issues in Methods. It is unacceptable that a number of citations were misrepresented. J. M. D. teaches Embryology and Anatomy to medical and graduate students at Georgetown University and serves as a consultant toxicologist for Exponent. In the past, he has consulted on cases involving potential developmental and reproductive outcomes after exposure to selective serotonin uptake inhibitors (SSRIs). He has no ongoing agreement with any organization involved in SSRI litigation. L. D. W. and L. A. D. have no potential conflicts of interest. Data sharing is not applicable to this article as no new data were created or analyzed in this study.

This article reports on a standard set of developmental and reproductive toxicity studies with molindone. I submit that the presented data does not support the major conclusion as stated in the Abstract (“Molindone was not developmentally toxic in rats or rabbits at 69X and 6X clinical exposures, confirming the reproductive safety of molindone”). The Discussion discounts effects on pup survival because they “did not follow a dose-dependent trend and did not result in any differences in mean litter size during lactation.” However, the effects at 20 and 40 mg/kg/day were essentially equal and do not have to be dose-related to be real, and only LD 1 litter size is reported. Lack of embryonic/fetal lethality in the EFD study does not negate a specific lethal effect on pups. The authors contradict their own conclusion by stating with no explanation “…while pup growth was significantly reduced at the high dose.” They then suggest hypoactivity could have reduced maternal care and indirectly caused reductions in pup survival. One must assume the reduced survivals at 20 and 40 mg/kg/day are drug-related developmental effects until a cross-fostering study or other evidence proves otherwise. Regarding the EFD toxicity study in rabbits with a high dose of 15 mg/kg/day (the 6X clinical exposure), there were increases in percent postimplantation loss at 10 and 15 mg/kg/day (2.9- and 3.2-fold above control) and decreased fetal weight at the high dose (8% below control); neither parameter statistically analyzed. Admittedly, the range-finding study did not have similar finding, but group sizes were very small. Neither the Results nor Discussion sections mentioned these changes. These indications of developmental toxicity are based only on presented data, but important sets of developmental data (i.e., fetal abnormalities and pup parameters) are not shown so readers are left to accept the authors' conclusions of “no treatment-related effects.” Given the oversights noted above, readers may rightly question conclusions of no effects in these parameters. Finally, contrary to the suggestion of no drug accumulation after repeated dosing, results in Table 3 show 24 of 30 groups with higher AUCs (up to 2.3-fold) at later times.

This article by Catlin et al. reports on well-designed EFDT studies in rats and rabbits. However, I would like to respectfully point out issues with the conclusions as stated in the abstract. I question the "exposure-related decreases in adjusted mean fetal weights per litter" (p. 888) in the lower dose groups Adjusted mean fetal weight (Tables 2 and 6) is not a routine parameter. I do not recall and cannot find this parameter in similar publications. So how it is calculated? Section 2.5 states "calculated by analysis of covariance in a manner similar to in-life adjusted maternal body weights, adjusting to the overall mean number of live fetuses…" The "dose-dependent increases in the incidences of ventricular septum defects and full supernumerary thoracolumbar ribs" in the lower dose groups are in no way definitive. In conclusion, virtually all conclusions regarding the lower dose groups of treated rats have been questioned. Additional information is needed to determine if VSDs are truly induced by vinpocetine. We should also look forward to a proposed mechanism to explain vinpocetine-induced embryo-fetal lethality. Finally, why was only a rabbit dose-range finding study reported when the online NTP 2016 annual report lists "Conventional teratology" for both rats and rabbit? There are no conflicts of interest.

GARDASIL®9, a 9-valent vaccine against human papillomavirus (9vHPV), was developed to prevent diseases mediated by HPV types 6/11/16/18/31/33/45/52/58. During the development of the vaccine, three nonclinical safety studies were conducted to evaluate repeat-dose toxicity and prenatal and postnatal developmental toxicity in Sprague-Dawley rats. In all studies, the vaccine was administered via intramuscular injections of 0.5 mL (the human dose) divided equally into each quadriceps muscle. In the repeat-dose toxicity study, potential local and systemic toxic effects of the 9vHPV vaccine were evaluated after 4 doses given 21 days apart and after a 21-day recovery period. In the prenatal study, virgin females were dosed at 5 and 2 weeks prior to mating and on Gestation Day [GD] 6 (3 total doses). Potential postnatal developmental toxicity of the vaccine formulation was evaluated after 4 total doses (premating to lactation). There were no treatment-related unscheduled deaths in any studies. In the 3-month repeat-dose toxicity study, no adverse effects in male or female rats were observed. Anticipated systemic effects representing immunological responses and local inflammatory reactions at the injection sites were noted in the vaccine-treated groups, with a trend toward recovery by the end of the 21-day recovery period. In the prenatal developmental toxicity study, there was no evidence of toxicity in females given the vaccine. There were no effects on fertility or reproductive performance of the parental females and no evidence of developmental toxicity. In the postnatal study, there was no evidence of toxicity in vaccine-treated females and no evidence of developmental toxicity based on standard postnatal parameters, including behavioral testing and reproductive performance. The vaccine induced antibody responses in all studies and vaccine-specific antibodies were detected in offspring in the developmental toxicity studies. These results support the favorable safety profile of GARDASIL®9.

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.

A developing organism exposed to a toxicant will have a response that ranges from none to severe (i.e., death or malformation). The response at a given dosage may be termed teratogenic (or developmental toxic) severity and is dependent on exposure conditions. Prenatal/embryo-fetal developmental (EFD) toxicity studies in rodents and rabbits are the most consistent and definitive assessments of teratogenic severity, and teratogenesis screening assays are best validated against their results. A formula is presented that estimates teratogenic severity for each group, including control, within an EFD study. The developmental components include embryonic/fetal death, malformations, variations, and mean fetal weight. The contribution of maternal toxicity is included with multiplication factors to adjust for the extent of mortality, maternal body weight change, and other parameters deemed important. The derivation of the formula to calculate teratogenic severity is described. Various EFD data sets from the literature are presented to highlight considerations to the calculation of the various components of the formula. Each score is compared to the concurrent control group to obtain a relative teratogenic severity. The limited studies presented suggest relative scores of two- to <fivefold higher than control have detectable but a low level of teratogenic severity, and scores ≥ fivefold higher than control have increasingly more severe teratogenicity. Such scores may help refine the concept of an exposure-based validation list for use by proponents of screening assays (Daston et al., 2014) by estimating the severity of "positive" exposures, or in other situations by defining the severity of a LOAEL (lowest observed adverse effect level).

Routinely, compounds are assessed by developmental and reproductive toxicology (DART) studies to evaluate the potential for drug-induced birth defects. High-throughput micro-CT images are being used to evaluate skeletal abnormalities due to its ability to provide high quality images of bone structures. Currently, these micro-CT images are visually inspected for skeletal abnormalities, which is a time and resource intensive process. To reduce the resources needed for skeletal evaluation, we developed image analysis strategies that allow for automatic segmentation of whole body CT images into individual bones and use structural variations of shape characteristics to classify bones as normal or abnormal. Extraction of various structures in the skull and torso were accomplished sequentially starting with skull bones and moving towards the neck, vertebrae, ribs, and limbs. A total of 17 skull bones/structures (supraoccipital, mandible, squamosals, zygomatics, etc.) and 20 torso structures (ribs, spine, humerus, femur, tibia, etc.) were identified and isolated using this algorithm. Next, we used geometrical (volume, length, width, etc.) and shape-based characteristics to identify bones lying outside the normal distribution of numbers, shapes and sizes to flag fetuses for potential abnormalities. We applied this tool to a test data set of 167 fetuses with verified skeletal abnormalities and received sensitivity of 0.959 and specificity of 0.805. This analysis platform allows for fully automated batch processing of images. Future work will include further development of the current platform to improve performance.

High-throughput micro-CT imaging has been used in our laboratory to evaluate fetal skeletal morphology in developmental toxicology studies. Currently, the volume-rendered skeletal images are visually inspected and observed abnormalities are reported for compounds in development. To improve the efficiency and reduce human error of the evaluation, we implemented a framework to automate the evaluation process. The framework starts by dividing the skull into regions of interest and then measuring various geometrical characteristics. Normal/abnormal classification on the bone segments is performed based on identifying statistical outliers. In pilot experiments using rabbit fetal skulls, the majority of the skeletal abnormalities can be detected successfully in this manner. However, there are shape-based abnormalities that are relatively subtle and thereby difficult to identify using the geometrical features. To address this problem, we introduced a model-based approach and applied this strategy on the squamosal bone. We will provide details on this active shape model (ASM) strategy for the identification of squamosal abnormalities and show that this method improved the sensitivity of detecting squamosal-related abnormalities from 0.48 to 0.92.

Spike–wave discharges (SWDs) are thalamocortical oscillations that are often considered to be the EEG correlate of absence seizures. Genetic absence epilepsy rats of Strasbourg (GAERS) and Wistar Albino Glaxo rats from Rijswijk (WAG/Rij) exhibit SWDs and are considered to be genetic animal models of absence epilepsy. However, it has been reported that other rat strains have SWDs, suggesting that SWDs may vary in their prevalence, but all rats have a predisposition for them. This is important because many of these rat strains are used to study temporal lobe epilepsy (TLE), where it is assumed that there is no seizure-like activity in controls. In the course of other studies using the Sprague–Dawley rat, a common rat strain for animal models of TLE, we found that approximately 19% of 2- to 3-month-old naive female Sprague–Dawley rats exhibited SWDs spontaneously during periods of behavioral arrest, which continued for months. Males exhibited SWDs only after 3 months of age, consistent with previous reports (Buzsáki et al., 1990). Housing in atypical lighting during early life appeared to facilitate the incidence of SWDs.Spike–wave discharges were often accompanied by behaviors similar to stage 1–2 limbic seizures. Therefore, additional analyses were made to address the similarity. We observed that the frequency of SWDs was similar to that of hippocampal theta rhythm during exploration for a given animal, typically 7–8 Hz. Therefore, activity in the frequency of theta rhythm that occurs during frozen behavior may not reflect seizures necessarily. Hippocampal recordings exhibited high frequency oscillations (> 250 Hz) during SWDs, suggesting that neuronal activity in the hippocampus occurs during SWDs, i.e., it is not a passive structure. The data also suggest that high frequency oscillations, if rhythmic, may reflect SWDs. We also confirmed that SWDs were present in a common animal model of TLE, the pilocarpine model, using female Sprague–Dawley rats. Therefore, damage and associated changes to thalamic, hippocampal, and cortical neurons do not prevent SWDs, at least in this animal model. The results suggest that it is possible that SWDs occur in rodent models of TLE and that investigators mistakenly assume that they are stage 1–2 limbic seizures. We discuss the implications of the results and ways to avoid the potential problems associated with SWDs in animal models of TLE.

Preclinical imaging technologies are increasingly being applied to developmental toxicology studies in drug development to determine potential compound toxicity. Although most of these studies are conducted in a non-regulatory setting, there is interest in performing these imaging studies under applicable regulations, for example Good Laboratory Practices (GLP), to support regulatory decisions concerning drug safety. This manuscript will describe regulations and processes to consider when bringing an imaging technology into GLP compliance.