Toxicological assessment of lower alkyl methacrylate esters by a category approach.

Categories and read-across are essential tools for supplying information for assessments of endpoints without data while minimizing animal testing. This study is based on the guidance of ECHA in its Read-Across Framework (RAAF). A category of C1 - C8 alkyl methacrylate esters (methyl, ethyl, n-butyl, iso-butyl and 2-ethylhexyl) was constructed to fill in missing information for human health endpoints using read-across as a permitted adaptation under EU REACH. The esters form a series with common functional groups, small incremental changes of electrophilicity by molecular weight, and rapid hydrolysis by ester cleavage. Read-across is justified by two common specific modes of action, direct electrophilic reaction of the parent compounds and the potential inherent toxicities of the common metabolites methacrylic acid and the corresponding alcohols. The toxicological profile is very similar for all category members and not driven by the alcohol metabolites. Data gaps can be filled in with high confidence based on the number of studies available, the effects therein observed and the toxicological profiles of the hydrolysis products. The guidance provided by the RAAF enabled data gaps to be filled in a robust manner.

An Adverse Outcome Pathway (AOP) for forestomach tumors induced by non-genotoxic initiating events.

The utility of rodent forestomach tumor data for hazard and risk assessment has been examined for decades because humans do not have a forestomach, and these tumors occur by varying modes of action (MOAs). We have used the MOA for ethyl acrylate (EA) to develop an Adverse Outcome Pathway (AOP) for forestomach tumors caused by non-genotoxic initiating events. These tumors occur secondary to site of contact induced epithelial cytotoxicity and regenerative repair-driven proliferation. For EA, the critical initiating event (IE) is epithelial cytotoxicity, and supporting key events (KEs) at the cellular and tissue level are increased cell proliferation (KE1) resulting in sustained hyperplasia (KE2), with the adverse outcome of forestomach papillomas and carcinomas. For EA, a pre-molecular initiating event (pre-MIE) of sustained glutathione depletion is probable. Supporting data from butylated hydroxyanisole (BHA) are also reviewed. Although there may be some variability in the pre-MIEs and IEs for BHA and EA, they share the same KEs, and evidence for BHA confers support for the AOP. Evolved Bradford Hill considerations of biological plausibility, essentiality, and empirical support were evaluated per OECD guidance. Although an MIE is not specifically described, overall confidence in the AOP is high due to well-developed and accepted evidence streams, and the AOP can be used for regulatory applications including hazard identification and risk assessment for chemicals that act by this AOP.

Assessment of the mode of action underlying development of forestomach tumors in rodents following oral exposure to ethyl acrylate and relevance to humans.

Chronic repeated gavage dosing of high concentrations of ethyl acrylate (EA) causes forestomach tumors in rats and mice. For two decades, there has been general consensus that these tumors are unique to rodents because of: i) lack of carcinogenicity in other organs, ii) specificity to the forestomach (an organ unique to rodents which humans do not possess), iii) lack of carcinogenicity by other routes of exposure, and iv) obvious site of contact toxicity at carcinogenic doses. In 1986, EA was classified as possibly carcinogenic to humans by the International Agency for Research on Cancer (IARC). However, by applying a MOA analyses and human relevance framework assessment, the weight-of-evidence supports a cytotoxic MOA with the following key events: i) bolus delivery of EA to forestomach lumen and subsequent absorption, ii) cytotoxicity likely due to saturation of enzymatic detoxification, iii) chronic regenerative hyperplasia, and iv) spontaneous mutation due to increased cell replication and cell population. Clonal expansion of initiated cells thus results in late onset tumorigenesis. The key events in this 'wound and healing' MOA provide high confidence in the MOA as assessed by evolved Bradford-Hill Criteria. The weight-of-evidence supported by the proposed MOA, combined with a unique tissue that does not exist in humans, indicates that EA is highly unlikely to pose a human cancer hazard.

Mode-of-action and human relevance framework analysis for rat Leydig cell tumors associated with sulfoxaflor.

Sulfoxaflor, a molecule that targets sap-feeding insects, was assessed for carcinogenic potential in groups of 50 Fischer rats fed with diets containing 0, 25, 100, 500 (males), or 750 (females) ppm sulfoxaflor for 2 years according to OECD 453. Sulfoxaflor did not alter the number of rats with Leydig cell tumors (LCTs: 88% of controls and 90-92% in treated groups). The size of LCT was increased at 100 and 500 ppm. The spontaneous incidence of LCT in Fischer rat is 75-100% compared with less than 0.01% in humans. These fundamental interspecies differences in spontaneous incidence of LCT are the result of quantitative and qualitative differences in Leydig cell response to hormonal stimuli. There are nine known modes of actions (MoA) for LCT induction. Analysis sulfoxaflor data suggested a hormone-based dopamine enhancement MoA causing the LCT effect through: 1) increased neuronal dopamine release via specific dopaminergic neuron-based nicotinic acetylcholine receptor (nAChR) agonism, leading to 2) decreased serum prolactin (Prl) levels, 3) downregulation of luteinizing hormone receptor (LHR) gene expression in Leydig cells, 4) transient decreases in serum testosterone, 5) increased serum LH levels, and 6) promotion of LCTs. The analysis suggested that sulfoxaflor promoted LCTs through a subtle stimulation of dopamine release. The MoA for LCT promotion in the carcinogenicity study is considered to have no relevance to humans due to qualitative and quantitative differences between rat and human Leydig cells. Therefore, the Fischer 344 rat LCT promotion associated with lifetime administration of high-dose levels of sulfoxaflor would not pose a cancer hazard to humans.

Human relevance framework evaluation of a novel rat developmental toxicity mode of action induced by sulfoxaflor.

Sulfoxaflor (CAS# 946578-00-3) is a novel active substance with insecticidal properties mediated via its agonism on the highly abundant insect nicotinic acetylcholine receptor (nAChR). In developmental and reproductive toxicity studies, gestational exposure caused fetal abnormalities (primarily limb contractures) and reduced neonatal survival in rats, but not rabbits, following high-dose dietary exposure. Sulfoxaflor induced these effects via a novel mode of action (MoA) mediated by the fetal-type muscle nAChR with the following key events: (1) binding to the receptor, (2) agonism on the receptor, causing (3) sustained muscle contracture in the near-term fetus and neonatal offspring. This sustained muscle contracture results in misshapen limbs, bent clavicles, and reduced diaphragm function, which compromises respiration in neonatal rats at birth, reducing their survival. This review evaluates the weight of evidence for this MoA based upon the Bradford Hill criteria, includes a cross-comparison of applied and internal doses in a variety of in vitro, ex vivo, and in vivo study designs, examines alternative MoAs, and applies a Human relevance framework (HRF) to ascertain human risk for this rat MoA. The review indicated, with a high level of confidence, that the sulfoxaflor-induced fetal abnormalities and neonatal death in rats occur via a single MoA comprising sustained activation of the rat fetal-type muscle nAChR resulting in a sustained muscle contracture. This MoA is considered not relevant to humans, given fundamental qualitative differences in sulfoxaflor agonism on the rat versus the human muscle nAChR. Specifically, sulfoxaflor does not cause agonism on either the human fetal- or adult-type muscle nAChR.

Assessment of diurnal systemic dose of agrochemicals in regulatory toxicity testing--an integrated approach without additional animal use.

Integrated toxicokinetics (TK) data provide information on the rate, extent and duration of systemic exposure across doses, species, strains, gender, and life stages within a toxicology program. While routine for pharmaceuticals, TK assessments of non-pharmaceuticals are still relatively rare, and have never before been included in a full range of guideline studies for a new agrochemical. In order to better understand the relationship between diurnal systemic dose (AUC(24h)) and toxicity of agrochemicals, TK analyses in the study animals is now included in all short- (excluding acute), medium- and long-term guideline mammalian toxicity studies including reproduction/developmental tests. This paper describes a detailed procedure for the implementation of TK in short-, medium- and long-term regulatory toxicity studies, without the use of satellite animals, conducted on three agrochemicals (X11422208, 2,4-D and X574175). In these studies, kinetically-derived maximum doses (KMD) from short-term studies instead of, or along with, maximum tolerated doses (MTD) were used for the selection of the high dose in subsequent longer-term studies. In addition to leveraging TK data to guide dose level selection, the integrated program was also used to select the most appropriate method of oral administration (i.e., gavage versus dietary) of test materials for rat and rabbit developmental toxicity studies. The integrated TK data obtained across toxicity studies (without the use of additional/satellite animals) provided data critical to understanding differences in response across doses, species, strains, sexes, and life stages. Such data should also be useful in mode of action studies and to improve human risk assessments.

Screening and prioritization of nano- and microplastic particle toxicity studies for evaluating human health risks - development and application of a toxicity study assessment tool.

Concern regarding the human health implications that exposure to nano- and microplastic particles (NMPs) potentially represents is increasing. While there have been several years of research reporting on the ecotoxicological effects of NMPs, human health toxicology studies have only recently emerged. The available human health hazard data are thus limited, with potential concern regarding the relevance and reliability for understanding the potential human health implications. In this study we develop and apply a NMP toxicity screening assessment tool (NMP-TSAT) for evaluating human health effects studies against a suite of quality assurance and quality control (QA/QC) criteria for both in vivo and in vitro studies. A total of 74 studies representing either inhalation or oral exposure pathways were identified and evaluated. Assessment categories include particle characterization, experimental design, and applicability for risk assessment; with critical and non-critical criteria organized to allow screening and prioritization. It is observed that the majority of studies evaluated using the NMP-TSAT have been performed on monodisperse particles, predominately spheres (≈60%), consisting of polystyrene (≈46%). The majority of studies have tested particles < 5 µm, with a minimal particle size of 10 nm and a maximum particle size of about 200 µm. The total assessment score (TAS) possible for in vivo studies is 52, whereas for in vitro studies it is 46, which is based on receiving a maximum score of 2 against 26 and 23 criteria, respectively. The evaluated TAS ranged from between 12 and 44 and 16-34, for in vivo and in vitro studies, respectively. Given the challenges associated with prioritizing studies based on ranking them according to their TAS we propose a Tiered approach, whereby studies are initially screened based on how they score against various critical criteria, which have been defined for their relevance for assessing the hazards and risks for human health. In this instance, studies that score a minimum of '1' against each of the critical criteria, regardless of how they rank according to their TAS, are prioritized as part of a Tier 1 screening and prioritization phase, which would then be followed by an expert evaluation, representing a Tier 2 level of assessment. Using this approach we identify 10 oral ingestion and 2 inhalation studies that score at least 1 against all critical criteria. Lastly, several key observations for strengthening future effects studies are identified, these include a need for the generation and access to standard reference materials representative of human exposure to NMPs for use in toxicity test systems and/or the improved characterization and verification of test particle characteristics, and the adoption of study design guidance, such as recommended by OECD, when conducting either in vivo inhalation or oral ingestion toxicity tests. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s43591-021-00023-x.

Epigenetic screening in product safety assessment: are we there yet?

There has been a growing concern that epigenetic events, that is, heritable changes in gene expression superimposed on DNA nucleotide sequences, may be involved in chemically and/or nutritionally mediated adverse health outcomes, such as reproductive toxicity and cancer. This concern has been driven by an increasing number of studies reporting toxicant-induced alterations to the epigenome in the form of changes in DNA methylation, histone/chromatin remodeling, and altered expression of non-coding RNAs. These three major mechanisms of epigenetic modifications may have coordinated, independent, or potentially antagonistic influences on gene expression. Complicating this understanding is the incomplete understanding of the normal state and dynamic variation of the epigenome, which differs widely between cells, tissues, developmental state, age, strain, and species. This review serves as a framework to outline characteristics composing an ideal epigenetic screen(s) for hazard identification in product safety assessment. In order to implement such a screen, first there needs to be a better understanding of adaptive versus adverse changes in the epigenome, which includes identification of robust and reproducible causal links between epigenetic changes and adverse apical end points, and second development of improved reporter assay tools to monitor such changes. An ideal screen would be in vitro-based, medium- to high-throughput, and assess all three branches of epigenome control (i.e. methylation, histone modifications, non-coding RNAs), although also being quantitative, objective, portable (i.e. lab to lab), and relevant to humans.

Epigenetics and chemical safety assessment.

Epigenetics, as it pertains to biology and toxicology, can be defined as heritable changes in gene expression that do not involve mutations and are propagated without continued stimulus. Although potentially reversible, these heritable changes may be classified as mitotic, meiotic, or transgenerational, implicating the wide-ranging impact of epigenetic control in cellular function. A number of biological responses have been classified as being caused by an "epigenetic alteration," sometimes based on sound scientific evidence and often in lieu of an identified genetic mutation. Complicating the understanding and interpretation of perceived epigenetic alterations is an incomplete understanding of the normal state and dynamic variation of the epigenome, which can differ widely between cell and tissue types and stage of development or age. This emerging field is likely to have a profound impact on the study and practice of toxicology in coming years. This document reviews the current state of the science in epigenetic modifications, techniques used to measure these changes, and evaluates the current toxicology testing battery with respect to strengths and potential weaknesses in the identification of epigenetics changes. In addition, case studies implicating transgenerational effects induced by diethylstilbestrol, vinclozolin, and bisphenol A were reviewed to illustrate the application of epigenetics in safety assessment and the strengths and limitations of the study designs. An assessment of toxicology tests currently used in safety evaluation revealed that these tests are expected to identify any potential adverse outcomes resulting from epigenetic changes. Furthermore, in order to increase our understanding of the science of epigenetics in toxicology, this review has revealed that a solid understanding of the biology and variation in the epigenome is essential to contextualize concerns about possible adverse health effects related to epigenetic changes. Finally, the fundamental principles guiding toxicology studies, including relevant doses, dose-rates, routes of exposure, and experimental models, need to be taken into consideration in the design and interpretation of studies within this emerging area of science.

Whole embryo culture: a "New" technique that enabled decades of mechanistic discoveries.

Denis New's development of the rodent whole embryo culture (WEC) method in the early 1960s was a groundbreaking achievement that gave embryologists and teratologists an unprecedented degree of access to the developing postimplantation rodent embryo. In the five decades since its development, WEC has enabled detailed investigations into the regulation of normal embryo development as well as a plethora of research on mechanisms of teratogenesis as induced by a wide range of agents. In addition, WEC is one of the few techniques that has been validated for use in teratogenicity screening of drugs and chemicals. In this review, we retrace the steps leading to New's development of WEC, and highlight many examples in which WEC played a crucial role leading to important discoveries in teratological research. The impact of WEC on the field of teratology has been enormous, and it is anticipated that WEC will remain a preferred tool for teratologists and embryologists seeking to interrogate embryo development for many years to come.

Altered health outcomes in adult offspring of Sprague Dawley and Wistar rats undernourished during early or late pregnancy.

BACKGROUND: Birth weight in humans has been inversely associated with adult disease risk. Results of animal studies have varied depending on species, strain, and treatment. METHODS: We compared birth weight and adult health in offspring following 50% maternal undernutrition on gestation days (GD) 1-15 (UN1-15) or GD 10-21 (UN10-21) in Sprague Dawley and Wistar rats. Offspring from food-deprived dams were weighed and cross-fostered to control dams. Litters were weighed during lactation and initiating at weaning males were fed either control or a high-fat diet. Young and mature adult offspring were evaluated for obesity, blood pressure (BP), insulin response to oral glucose, and serum lipids. Nephron endowment, renal glucocorticoid receptor, and renin-aldosterone-angiotensin system components were measured. RESULTS: The UN10-21 groups had birth weights lower than controls and transient catch up growth by weaning. Neither strain demonstrated obesity or dyslipidemia following prenatal undernutrition, but long-term body weight deficits occurred in the UN groups of both strains. High-fat diet fed offspring gained more weight than control offspring without an effect of prenatal nutrition. Sprague Dawley were slightly more susceptible than Wistar rats to altered insulin response and increased BP following gestational undernutrition. Nephron endowment in Sprague Dawley but not Wistar offspring was lower in the UN10-21 groups. Glucocorticoid and renin-aldosterone-angiotensin system pathways were not altered. CONCLUSIONS: The most consistent effect of maternal undernutrition was elevated BP in offspring. Long-term health effects occurred with undernutrition during either window, but the UN10-21 period resulted in lower birth weight and more severe adult health effects.

The effects of marginal maternal vitamin A status on penta-brominated diphenyl ether mixture-induced alterations in maternal and conceptal vitamin A and fetal development in the Sprague Dawley rat.

BACKGROUND: Polybrominated diphenyl ether (PBDE) toxicity in rodents can be associated with disruptions in endocrine signaling. We previously reported that the penta-BDE mixture, DE-71, disrupts thyroid hormones and vitamin A metabolism in rats during lactation, and that this disruption is amplified in animals fed diets marginal in vitamin A. The ability of the DE-71 to disrupt vitamin A metabolism during the prenatal period has not been evaluated. While penta-BDE mixtures are not strong teratogens in pregnant animals fed standard commercial laboratory diets, we hypothesized that they could be teratogenic under conditions of marginal vitamin A status. METHODS: rats were fed diets containing 0.4 retinyl equivalents (RE, marginal) or 4.0 RE (adequate) of vitamin A per gram of diet. Pregnant animals were exposed to DE-71 (0, 6, 18, 60, or 120 mg/kg) from gestation days (GD) 6-11.5, or on GD 6-19.5. RESULTS: DE-71 treatment resulted in dose-responsive reductions in maternal thyroid hormone and markers of vitamin A metabolism, with the latter reduction amplified in marginal vitamin A dams. Fetuses from marginal vitamin A, DE-71-exposed dams exhibited a dose-responsive increase in liver retinol binding protein levels. DE-71 treatment did not result in gross malformations; however, consistent with our hypothesis, GD 20 fetal weights were lower, and skeletal ossification was less when DE-71 exposure occurred concomitant with a marginal vitamin A status. For several endpoints, observable effects were evident at the lowest dose tested, consistent with a dose-response trend. CONCLUSIONS: The results of this study support the concept that marginal vitamin A status enhances the disruptive effects of DE-71 during prenatal development.

Systems Modeling of Developmental Vascular Toxicity.

The more than 80,000 chemicals in commerce present a challenge for hazard assessments that toxicity testing in the 21st century strives to address through high-throughput screening (HTS) assays. Assessing chemical effects on human development adds an additional layer of complexity to the screening, with a need to capture complex and dynamic events essential for proper embryo-fetal development. HTS data from ToxCast/Tox21 informs systems toxicology models, which incorporate molecular targets and biological pathways into mechanistic models describing the effects of chemicals on human cells, 3D organotypic culture models, and small model organisms. Adverse Outcome Pathways (AOPs) provide a useful framework for integrating the evidence derived from these in silico and in vitro systems to inform chemical hazard characterization. To illustrate this formulation, we have built an AOP for developmental toxicity through a mode of action linked to embryonic vascular disruption (Aop43). Here, we review the model for quantitative prediction of developmental vascular toxicity from ToxCast HTS data and compare the HTS results to functional vascular development assays in complex cell systems, virtual tissues, and small model organisms. ToxCast HTS predictions from several published and unpublished assays covering different aspects of the angiogenic cycle were generated for a test set of 38 chemicals representing a range of putative vascular disrupting compounds (pVDCs). Results boost confidence in the capacity to predict adverse developmental outcomes from HTS in vitro data and model computational dynamics for in silico reconstruction of developmental systems biology. Finally, we demonstrate the integration of the AOP and developmental systems toxicology to investigate the unique modes of action of two angiogenesis inhibitors.

In vitro genotoxicity studies: n-Butyl acrylate L5178Y mouse lymphoma (TK+/- locus assay), 2-Ethylhexyl acrylate gene mutation assay in Chinese hamster V79 cells, and 2-Ethylhexyl acrylate micronucleus test in human lymphocytes.

Available point mutation tests have shown inconsistent results with various acrylates. Most of those tests were performed prior to OECD guidelines and appropriate data regarding cytotoxicity are not given. Data from three current OECD guideline compliant experiments conducted under GLP are provided. They include (a) an in vitro mouse lymphoma (TK+/-) assay (OECD 490) [3], (b) an in vitro HPRT locus gene mutation assay utilizing cultures of Chinese hamster V79 cells (OECD 476) [1], and (c) an in vitro micronucleus test in human lymphocytes (OECD 487) [2]. Test materials were not mutagenic under these experimental conditions, adding to the weight-of-evidence of non-genotoxicity for this group of chemicals.

Critical evaluation of 2-ethylhexyl acrylate dermal carcinogenicity studies using contemporary criteria.

Skin tumors have been observed in C3H/HeJ mice following treatment with high and strongly irritating concentrations of 2-ethylhexyl acrylate (2-EHA). Dermal carcinogenicity studies performed with 2-EHA are reviewed, contrasting the results in two mouse strains (C3H/HeJ and NMRI) under different dosing regimens. Application of contemporary evaluation criteria to the existing dermal carcinogenicity dataset demonstrates that 2-EHA induces skin tumors only at concentrations exceeding an maximum tolerated dose (MTD) and in the immune-dysregulated C3H/HeJ mouse model. Overall, the available chronic toxicity and genotoxicity data on 2-EHA support a non-genotoxic chemical irritant mechanism, whereby chronic irritation leads to inflammation, tissue injury, and wound repair, the latter of which is disrupted in C3H/HeJ mice and leads to tumor formation. Tumor response information in excess of an MTD should not be considered in a human hazard or risk assessment paradigm. For the purposes of an appropriate hazard assessment, 2-EHA did not cause or initiate dermal carcinogenesis in an immune competent (NMRI) mouse model, and, even in the immune compromised C3H/HeJ model, did not induce skin tumors at doses which did not exceed the MTD.

A review of the genotoxic, mutagenic, and carcinogenic potentials of several lower acrylates.

Lower alkyl acrylate monomers include methyl-, ethyl-, n-butyl-, and 2-ethylhexyl acrylate. These acrylates are used in the manufacture of acrylic polymers and copolymers for plastics, food packaging, adhesives, and cosmetic formulations. Although there is limited potential for human environmental exposure, occupational exposure can occur via inhalation and dermal contact. Recently, new genotoxicity data have been generated, along with in silico and in vitro read-cross analyses, for these acrylates. The availability of high-throughput screening (HTS) data through the ToxCast™/Tox21 databases allows for consideration of computational toxicology and organization of these data according to the ten key characteristics of carcinogens. Therefore, we conducted a comprehensive review to evaluate the mechanistic, toxicokinetic, animal, and human data, including HTS data, for characterizing the potential carcinogenicity, mutagenicity, and genotoxicity of these acrylates. Toxicokinetic data demonstrate that these acrylates are metabolized rapidly by carboxylesterase hydrolysis and conjugation with glutathione. HTS data demonstrated an overall lack of bioactivity in cancer-related pathways. Overall, the genotoxicity and mutagenicity data support a cytotoxic, non-genotoxic mechanism for these acrylates. Cancer bioassay studies conducted by the oral, dermal, and inhalation routes in animal models with these acrylates did not show any increase in tumor incidence, with two exceptions. At high doses, and secondary to chronic site-of-contact irritation and corrosion, rodent forestomach tumors were induced by oral gavage dosing with ethyl acrylate, and skin tumors were observed following chronic dermal dosing with 2-ethylhexyl acrylate in C3H/HeJ inbred mice (a strain with deficiencies in wound healing), but not in the outbred NMRI strain. For both dermal and forestomach cancers, tumorigenesis is secondary to high doses and long-term tissue damage, shown to be reversible. With evidence that these chemicals are not genotoxic, and that they cause forestomach and dermal tumors through chronic irritation and regenerative proliferation mechanisms, these acrylates are unlikely to pose a human cancer hazard.

Embryonic vascular disruption adverse outcomes: Linking high throughput signaling signatures with functional consequences.

Embryonic vascular disruption is an important adverse outcome pathway (AOP) as chemical disruption of cardiovascular development induces broad prenatal defects. High throughput screening (HTS) assays aid AOP development although linking in vitro data to in vivo apical endpoints remains challenging. This study evaluated two anti-angiogenic agents, 5HPP-33 and TNP-470, across the ToxCastDB HTS assay platform and anchored the results to complex in vitro functional assays: the rat aortic explant assay (AEA), rat whole embryo culture (WEC), and the zebrafish embryotoxicity (ZET) assay. Both were identified as putative vascular disruptive compounds (pVDCs) in ToxCastDB and disrupted angiogenesis and embryogenesis in the functional assays. Differences were observed in potency and adverse effects: 5HPP-33 was embryolethal (WEC and ZET); TNP-470 produced caudal defects at lower concentrations. This study demonstrates how a tiered approach using HTS signatures and complex functional in vitro assays might be used to prioritize further in vivo developmental toxicity testing.

Embryonic vascular disruption adverse outcomes: Linking high-throughput signaling signatures with functional consequences.

Embryonic vascular disruption is an important adverse outcome pathway (AOP) as chemical disruption of cardiovascular development induces broad prenatal defects. High-throughput screening (HTS) assays aid AOP development although linking in vitro data to in vivo apical endpoints remains challenging. This study evaluated two anti-angiogenic agents, 5HPP-33 and TNP-470, across the ToxCastDB HTS assay platform and anchored the results to complex in vitro functional assays: the rat aortic explant assay (AEA), rat whole embryo culture (WEC), and the zebrafish embryotoxicity (ZET) assay. Both were identified as putative vascular disruptive compounds (pVDCs) in ToxCastDB and disrupted angiogenesis and embryogenesis in the functional assays. Differences were observed in potency and adverse effects: 5HPP-33 was embryolethal (WEC and ZET); TNP-470 produced caudal defects at lower concentrations. This study demonstrates how a tiered approach using HTS signatures and complex functional in vitro assays might be used to prioritize further in vivo developmental toxicity testing.

Dietary Route of Exposure for Rabbit Developmental Toxicity Studies.

Dietary administration is a relevant route of oral exposure for regulatory toxicity studies of agrochemicals as it mimics potential human intake of the chemical via treated crops and commodities. Moreover, dietary administration of test compounds during a developmental toxicity study can deliver a prolonged and stable systemic exposure to the embryo or fetus at all stages of development. In this study, strategies were employed to optimize rabbit test material consumption via diet. Comparative toxicokinetic profiles of gavage versus dietary administration were evaluated in pregnant or non-pregnant New Zealand White rabbits for 2 novel agrochemicals with different plasma half-lives of elimination (sulfoxaflor, t½ = 13.5 h and halauxifen, t½ = 1 h). Dietary administration of sulfoxaflor resulted in stable 24-h plasma concentrations, whereas gavage administration resulted in a 3-fold fluctuation in plasma levels between Cmax and Cmin Dietary administration of sulfoxaflor resulted in a 2-fold higher nominal and diurnal systemic dose when compared with gavage dosing due to Cmax-related maternal toxicity following gavage. Results with the shorter half-life molecule, halauxifen, were more striking with a 6-fold diurnal fluctuation by the dietary route compared with a 368-fold fluctuation between Cmax and Cmin by gavage. Furthermore, plasma halauxifen was detectable only up to 12 h following gavage but up to 24 h following dietary administration. Finally, the presence of these compounds in fetal blood samples was demonstrated, confirming that dietary exposure is appropriate for achieving fetal exposure. Collectively, the results of these studies support the use of dietary exposure in rabbit developmental toxicity studies.

Disposition of glycolic acid into rat and rabbit embryos in vitro.

High dose gavage administration of ethylene glycol (EG) induces teratogenicity in rodents, but not in rabbits, resulting from saturation of intermediate EG metabolism and glycolic acid (GA) accumulation. In vivo, rat embryos sequester GA 2-4-fold higher than maternal blood, a phenomenon absent in rabbits and proposed not to occur in humans. This research explored the mechanisms of GA disposition into rat and rabbit conceptuses using whole embryo culture (WEC). Rat and rabbit embryos concentrated GA from the culture medium. In vitro to in vivo discordance in the rabbit plausibly stemmed from anatomical differences between these models. GA sequestration was attenuated at 4°C in both species. Rat embryos further demonstrated pH-dependence of GA sequestration and inhibition by D-lactic acid. These data suggest GA disposition into rat and rabbit embryos is energy- and pH-dependent, and carrier-mediated. Anatomical and maternal-to-conceptal pH gradient differences likely underlie the lack of enhanced GA disposition in non-rodent species.