Addressing the relevance of polystyrene nano- and microplastic particles used to support exposure, toxicity and risk assessment: implications and recommendations.

Background: There has been an exponential increase in the number of studies reporting on the toxicological effects associated with exposure to nano and microplastic particles (NMPs). The majority of these studies, however, have used monodispersed polystyrene microspheres (PSMs) as 'model' particles. Here we review the differences between the manufacture and resulting physicochemical properties of polystyrene used in commerce and the PSMs most commonly used in toxicity studies.

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

Unlabelled: 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.

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 21 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.

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.

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.

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).

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.

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.

The role of ethyl acrylate induced GSH depletion in the rodent forestomach and its impact on MTD and in vivo genotoxicity in developing an adverse outcome pathway (AOP).

Adverse outcome pathways (AOP) and mode of action (MOA) frameworks help evaluate the toxicity findings of animal studies and their relevance to humans. To effectively use these tools to improve hazard identification and risk assessments for ethyl acrylate (EA), knowledge gaps in metabolism and genotoxicity were identified and addressed. For EA, hypothesized early key events relate to its irritation potential: concentration dependent irritation and cytotoxicity, progressing to regenerative proliferation and forestomach carcinogenicity after repeated oral bolus application in rodents.

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.

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.

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.

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.

Distinguishing between maternally-mediated and directly embryotoxic modes-of-action for postimplantation loss induced in rats by 2-amino-2-methylpropanol.

In rats, 2-amino-2-methylpropanol (AMP) caused an increase in postimplantation loss in an oral reproductive/developmental toxicity screening assay but not in a dermal developmental toxicity assay. Studies were performed to characterize the mode of action and determine whether the postimplantation loss was a result of direct embryotoxicity or a maternally mediated effect. The studies identified that the postimplantation loss occurs shortly after implantation, has a steep dose response with a clear threshold, requires exposure to AMP for a period of approximately 2-3 weeks prior to gestation and does not involve direct embryo toxicity.

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.

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.

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).

Elevated blood pressure in offspring of rats exposed to diverse chemicals during pregnancy.

Adverse intrauterine environments have been associated with increased risk of later cardiovascular disease and hypertension. In an animal model using diverse developmental toxicants, we measured blood pressure (BP), renal nephron endowment, renal glucocorticoid receptor (GR) gene expression, and serum aldosterone in offspring of pregnant Sprague Dawley rats exposed to dexamethasone (Dex), perfluorooctane sulfonate (PFOS), atrazine, perfluorononanoic acid (PFNA), arsenic, or nicotine. BP was assessed by tail cuff photoplethysmography, nephron endowment by confocal microscopy, and renal GR mRNA by qPCR.

A novel mode-of-action mediated by the fetal muscle nicotinic acetylcholine receptor resulting in developmental toxicity in rats.

Sulfoxaflor (X11422208), a novel agricultural molecule, induced fetal effects (forelimb flexure, hindlimb rotation, and bent clavicle) and neonatal death in rats at high doses (≥ 400 ppm in diet); however, no such effects occurred in rabbit dietary studies despite achieving similar maternal and fetal plasma exposure levels. Mode-of-action (MoA) studies were conducted to test the hypothesis that the effects in rats had a single MoA induced by sulfoxaflor agonism on the fetal rat muscle nicotinic acetylcholine receptor (nAChR). The studies included cross-fostering and critical windows of exposure studies in rats, fetal ((α1)(2)β1γδ) and adult ((α1)(2)β1δε) rat and human muscle nAChR in vitro agonism experiments, and neonatal rat phrenic nerve-hemidiaphragm contracture studies.

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.

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.

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.

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.

The influence of gestational zinc deficiency on the fetal insulin-like growth factor axis in the rat.

The insulin-like growth factor (IGF) axis, a key regulator of embryonic growth and development, is exquisitely sensitive to the nutrient status of the animal. In addition to macronutrient deficiencies, zinc deficiency can impact the IGF axis. Gestational zinc deficiency is teratogenic, resulting in intrauterine growth retardation and structural abnormalities.

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.