Investigating open access new approach methods (NAM) to assess biological points of departure: A case study with 4 neurotoxic pesticides

Abstract

Open access new approach methods (NAM) in the US EPA ToxCast program and NTP Integrated Chemical Environment (ICE) were used to investigate activities of four neurotoxic pesticides: endosulfan, fipronil, propyzamide and carbaryl. Concordance of in vivo regulatory points of departure (POD) adjusted for interspecies extrapolation (AdjPOD) to modelled human Administered Equivalent Dose (AED_Human) was assessed using 3-compartment or Adult/Fetal PBTK in vitro to in vivo extrapolation. Model inputs were from Tier 1 (High throughput transcriptomics: HTTr, high throughput phenotypic profiling: HTPP) and Tier 2 (single target: ToxCast) assays. HTTr identified gene expression signatures associated with potential neurotoxicity for endosulfan, propyzamide and carbaryl in non-neuronal MCF-7 and HepaRG cells. The HTPP assay in U-2 OS cells detected potent effects on DNA endpoints for endosulfan and carbaryl, and mitochondria with fipronil (propyzamide was inactive). The most potent ToxCast assays were concordant with specific components of each chemical mode of action (MOA). Predictive adult IVIVE models produced fold differences (FD) < 10 between the AED_Human and the measured in vivo AdjPOD. The 3-compartment model was concordant (i.e., smallest FD) for endosulfan, fipronil and carbaryl, and PBTK was concordant for propyzamide. The most potent AED_Human predictions for each chemical showed HTTr, HTPP and ToxCast were mainly concordant with in vivo AdjPODs but assays were less concordant with MOAs. This was likely due to the cell types used for testing and/or lack of metabolic capabilities and pathways available in vivo. The Fetal PBTK model had larger FDs than adult models and was less predictive overall.

Keywords: Carbaryl; Endosulfan; Fipronil; High Throughput Phenotypic Profile (HTPP); High Throughput Transcriptomics (HTTr); Integrated Chemical Environment (ICE); New Approach Methods (NAM); Propyzamide; ToxCast.

Graphical abstract

Fig. 1

Structures of the neurotoxic pesticides examined in this study.

Fig. 2

A tiered approach encompassing New Approach Methodologies (NAM) is described for screening, prioritizing and chemical characterization. Tier 1 involves knowledge of chemical structure, along with use of assays with broad coverage, high content, and multiple cell types. Chemicals with predictions of biological targets or pathways from Tier 1 can be further characterized in Tier 2 by use of in vitro targeted assays (e.g., ToxCast). Tier 1 High throughput transcriptomics (HTTr) gene expression signature and high throughput phenotypic profile endpoint (HTPP) benchmark doses (BMD µM) and Tier 2 ToxCast AC₅₀s (µM) can be used in various Integrated Chemical Environment Tools such as in vitro to in vivo extrapolation (IVIVE) models to produce human administered equivalent doses (AED_Human mg/kg) as points of departure (POD). Predicted PODs can be compared to regulatory in vivo PODs after applying an interspecies (animal to human) 10-fold extrapolation (in vivo AdjPOD), to assess concordance.

Fig. 3

Fold differences (FD) between AED_Human predictions for selected ENDO assays in the adult 3COMP and Fetal PBTK models and AdjLOEL in vivo regulatory POD. The assays are divided into HTTr, HTPP, and ToxCast (endocrine, neuronal activity, and metabolism). The FD are shown on each bar rounded to the nearest whole number.

Fig. 4

Fold differences (FD) between AED_Human predictions for selected FIP assays in the adult 3COMP and Fetal PBTK models and AdjBMD in vivo regulatory POD. The assays are divided into HTTr, HTPP, and ToxCast (endocrine, neuronal activity, and metabolism). The FD are shown on each bar rounded to the nearest whole number.

Fig. 5

Fold differences (FD) between AED_Human predictions for selected PRZ assays in the adult PBTK and Fetal PBTK models and AdjLOEL in vivo measured POD. The assays are divided into HTTr and ToxCast (metabolism). The FD are shown on each bar rounded to the nearest whole number.

Fig. 6

Fold differences between AED_Human predictions for selected PRZ assays in the 3COMP or Fetal PBTK models and AdjBMD in vivo regulatory POD. The assays are divided into HTTr, HTPP, and ToxCast (esterase, development, neuronal activity, and metabolism). The FD are shown on each bar rounded to the nearest whole number.