Identifying the landscape of developmental toxicity new approach methodologies.

BACKGROUND: The dynamics and complexities of in utero fetal development create significant challenges in transitioning from lab animal-centric developmental toxicity testing methods to assessment strategies based on new approach methodologies (NAMs). Nevertheless, considerable progress is being made, stimulated by increased research investments and scientific advances, such as induced pluripotent stem cell-derived models. To help identify developmental toxicity NAMs for toxicity screening and potential funding through the American Chemistry Council's Long-Range Research Initiative, a systematic literature review was conducted to better understand the current landscape of developmental toxicity NAMs. METHODS: Scoping review tools were used to systematically survey the literature (2010-2021; ~18,000 references identified), results and metadata were then extracted, and a user-friendly interactive dashboard was created. RESULTS: The data visualization dashboard, developed using Tableau® software, is provided as a free, open-access web tool. This dashboard enables straightforward interactive queries and visualizations to identify trends and to distinguish and understand areas or NAMs where research has been most, or least focused. CONCLUSIONS: Herein, we describe the approach and methods used, summarize the benefits and challenges of applying the systematic-review techniques, and highlight the types of questions and answers for which the dashboard can be used to explore the many different facets of developmental toxicity NAMs.

Current status and future directions for a neurotoxicity hazard assessment framework that integrates in silico approaches.

Neurotoxicology is the study of adverse effects on the structure or function of the developing or mature adult nervous system following exposure to chemical, biological, or physical agents. The development of more informative alternative methods to assess developmental (DNT) and adult (NT) neurotoxicity induced by xenobiotics is critically needed. The use of such alternative methods including in silico approaches that predict DNT or NT from chemical structure (e.g., statistical-based and expert rule-based systems) is ideally based on a comprehensive understanding of the relevant biological mechanisms. This paper discusses known mechanisms alongside the current state of the art in DNT/NT testing. In silico approaches available today that support the assessment of neurotoxicity based on knowledge of chemical structure are reviewed, and a conceptual framework for the integration of in silico methods with experimental information is presented. Establishing this framework is essential for the development of protocols, namely standardized approaches, to ensure that assessments of NT and DNT based on chemical structures are generated in a transparent, consistent, and defendable manner.

Characterization of dermal sensitization potential for industrial or agricultural chemicals with EpiSensA.

The regulatory community is transitioning to the use of nonanimal methods for dermal sensitization assessments; however, some in vitro assays have limitations in their domain of applicability depending on the properties of chemicals being tested. This study explored the utility of epidermal sensitization assay (EpiSensA) to evaluate the sensitization potential of complex and/or "difficult to test" chemicals. Assay performance was evaluated by testing a set of 20 test chemicals including 10 methacrylate esters, 5 silicone-based compounds, 3 crop protection formulations, and 2 surfactant mixtures; each had prior in vivo data plus some in silico and in vitro data. Using the weight of evidence (WoE) assessments by REACH Lead Registrants, 14 of these chemicals were sensitizers and, six were nonsensitizers based on in vivo studies (local lymph node assay [LLNA] and/or guinea pig studies). The EpiSensA correctly predicted 16/20 materials with three test materials as false positive and one silane as false negative. This silane, classified as weak sensitizer via LLNA, also gave a "false negative" result in the KeratinoSens™ assay. Overall, consistent with prior evaluations, the EpiSensA demonstrated an accuracy level of 80% relative to available in vivo WoE assessments. In addition, potency classification based on the concentration showing positive marker gene expression of EpiSensA was performed. The EpiSensA correctly predicted the potency for all seven sensitizing methacrylates classified as weak potency via LLNA (EC3 ≥ 10%). In summary, EpiSensA could identify dermal sensitization potential of these test substances and mixtures, and continues to show promise as an in vitro alternative method for dermal sensitization.

Pronamide: Weight of evidence for potential estrogen, androgen or thyroid effects.

Based on the exposure potential to humans and environment, pronamide was one of 52 chemicals on the first list evaluated under US EPA's Endocrine Disruptor Screening Program (EDSP). The purpose of EDSP is to screen chemicals for their potential to interact with estrogen-, androgen-, or thyroid-signaling pathways. A battery of 11 Tier 1 assays was completed for pronamide in accordance with EDSP test guidelines. In addition, Other Scientifically Relevant Information, which included existing data from regulatory guideline studies and published literature, was used in a weight-of-evidence (WoE) evaluation of potential endocrine activity. The WoE conclusion is that pronamide does not interact directly with estrogen, androgen, or thyroid receptors or post-receptor events. Across in vivo studies, the liver is consistently and reproducibly the target organ for pronamide's effects. Pronamide activates hepatocytic nuclear receptors (including constitutive androstane receptor), induces hepatic enzymes, produces hepatocellular hypertrophy and increases liver weights. These changes are coupled with increased metabolic activity and a subsequent increased metabolism and/or clearance of both steroid and thyroid hormones. Thus, while pronamide alters some endocrine-sensitive endpoints in EDSP Tier 1 assays, effects on liver metabolism likely explain altered hormone levels and indirect endocrine changes.

An F1-extended one-generation reproductive toxicity study in Crl:CD(SD) rats with 2,4-dichlorophenoxyacetic acid.

2,4-Dichlorophenoxyacetic acid (2,4-D) was assessed for systemic toxicity, reproductive toxicity, developmental neurotoxicity (DNT), developmental immunotoxicity (DIT), and endocrine toxicity. CD rats (27/sex/dose) were exposed to 0, 100, 300, 600 (female), or 800 (male) ppm 2,4-D in diet. Nonlinear toxicokinetic behavior was shown at high doses; the renal clearance saturation threshold for 2,4-D was exceeded markedly in females and slightly exceeded in males. Exposure was 4 weeks premating, 7 weeks postmating for P1 males and through lactation for P1 females. F1 offspring were examined for survival and development, and at weaning, pups were divided in cohorts, by sex and dose, and by systemic toxicity (10), DNT (10), DIT (20), and reproductive toxicity (≥ 23). Remaining weanlings were evaluated for systemic toxicity and neuropathology (10-12). Body weight decreased during lactation in high-dose P1 females and in F1 pups. Kidney was the primary target organ, with slight degeneration of proximal convoluted tubules observed in high-dose P1 males and in high-dose F1 males and females. A slight intergenerational difference in kidney toxicity was attributed to increased intake of 2,4-D in F1 offspring. Decreased weanling testes weights and delayed preputial separation in F1 males were attributed to decreased body weights. Endocrine-related effects were limited to slight thyroid hormone changes and adaptive histopathology in high-dose GD 17 dams seen only at a nonlinear toxicokinetic dose. 2,4-D did not cause reproductive toxicity, DNT, or DIT. The "No Observed Adverse Effect Level" for systemic toxicity was 300 ppm in both males (16.6 mg/kg/day) and females (20.6 mg/kg/day), which is approximately 6700- to 93 000-fold higher than that reported for 2,4-D exposures in human biomonitoring studies.

Interlaboratory study comparison of the 15-day intact adult male rat screening assay: evaluation of an antithyroid chemical and a negative control chemical.

Validation of the 15-day intact adult male rat screening assay (IAMRSA), an endocrine activity screen, was extended beyond the 28 substances evaluated to date. Two independent laboratories evaluated specificity using allyl alcohol (AA), a putative negative control, and DE-71 (technical grade pentabromodiphenyl ether) for comparison with previous pubertal assays that demonstrated thyroid effects. Male rats (15/group) were gavaged daily with AA (0, 10, 30, or 40 mg/kg/day) or DE-71 (0, 3, 30, or 60 mg/kg/day) for 15 days. Body and organ weights and serum hormone concentrations were measured, and a limited histopathological assessment was conducted. AA results were considered negative at doses that did not exceed the maximum tolerated dose (MTD); effects reported were dose-related decreases in weight gain, increased liver weights and, although the pattern varied across studies, alterations in some androgen-sensitive endpoints in the high-dose where the maximum tolerated dose was exceeded. In the DE-71 studies, dose-dependent increases in liver weights (consistent with hepatic enzyme induction), decreases in tri-iodothyronine and thyroxine, concomitant thyroid stimulating hormone increases were observed and one laboratory reported histopathological thyroid changes in mid- and high-dose groups, and the other increased thyroid weights. For DE-71, the IAMRSA was comparable in sensitivity to the pubertal assays. Overall, the specificity and sensitivity of the IAMRSA for deployment in an endocrine screening battery are supported. However, differentiating primary endocrine-mediated effects from secondary effects caused by systemic toxicity will be challenging, emphasizing the need to utilize a battery of assays and a weight of evidence approach when evaluating the potential endocrine activity of chemicals.

Dynamic changes in lipids and proteins of maternal, fetal, and pup blood and milk during perinatal development in CD and Wistar rats.

An understanding of the physiological factors that regulate perinatal dosimetry is essential to improve the ability of physiologically based (PB) pharmacokinetic (PK) models to predict chemical risks to children. However, the impact of changing maternal/offspring physiology on PK during gestation and lactation remains poorly understood. This research determined lipid and protein changes in blood, milk and amniotic fluid of CD and Wistar dams, fetuses and neonates to improve the precision of perinatal PBPK modeling. Samples were collected from time-mated CD dams, fetuses, and pups on gestation day (GD) 18 and 20 (sperm positive = GD 0) or lactation day 0 (day of birth), 1, 3, 5, 10, 15, and 20 (n > or = 5 per time point). Fewer time points were sampled in Wistar rats, which showed similar patterns to CDs. Relative to nonpregnant dams, maternal serum protein levels (albumin, total protein and globulin) each decreased by approximately 20% during late gestation, whereas maternal serum lipids (triglycerides, low density lipoproteins, and phospholipids) increased up to fourfold. These physiological changes can impact maternal PK of both protein-bound and lipophilic chemicals. During lactation, triglycerides in milk were greater than 100-fold higher than maternal serum, favoring the disposition of lipophilic chemicals into milk and potentially increasing neonatal rodent exposure during critical stages of postnatal development. Serum protein levels in pups were two- to threefold lower than adults at birth, which may increase the bioavailability of protein-bound compounds. These data will aid in the interpretation of perinatal toxicity studies and improve the accuracy of predictive perinatal PBPK models.

The effect of route, vehicle, and divided doses on the pharmacokinetics of chlorpyrifos and its metabolite trichloropyridinol in neonatal Sprague-Dawley rats.

There is a paucity of data on neonatal systemic exposure using different dosing paradigms. Male CD (Sprague-Dawley derived) rats at postnatal day (PND) 5 were dosed with chlorpyrifos (CPF, 1 mg/kg) using different routes of exposure, vehicles, and single versus divided doses. Blood concentrations of CPF and its primary metabolite, trichloropyridinol, were measured at multiple times through 24 h. Groups included were single gavage bolus versus divided gavage doses in corn oil (one vs. three times in 24h), single gavage bolus versus divided gavage doses in rat milk, and sc administration in dimethyl sulfoxide (DMSO). These data were compared with lactational exposure of PND 5 pups from dams exposed to CPF in the diet at 5 mg/kg/day for 4 weeks or published data from dams exposed to daily gavage with CPF at 5 mg/kg/day. Maternal blood CPF levels were an order of magnitude lower from dietary exposure than gavage (1.1 vs. 14.8 ng/g), and blood CPF levels in PND 5 pups that nursed dietary-exposed or gavage-exposed dams were below the limit of detection. Single gavage doses of 1 mg/kg CPF in corn oil vehicle in pups resulted in CPF blood levels of 49 ng/g and in milk vehicle about 9 ng/g. Divided doses led to lower peak CPF levels. A bolus dose of 1 mg/kg CPF in DMSO administered sc appeared to have substantially altered pharmacokinetics from orally administered CPF. To be meaningful for risk assessment, neonatal studies require attention to the exposure scenario, since route, vehicle, dose, and frequency of administration result in different systemic exposure to the test chemical and its metabolites.