Skeletal teratogenicity of industrial and environmental chemicals predicted with human pluripotent stem cells in vitro

EPA Grant Number: R839502
Title: Skeletal teratogenicity of industrial and environmental chemicals predicted with human pluripotent stem cells in vitro
Investigators: zur Nieden, Nicole I , Volz, David C.
Institution: University of California - Riverside
EPA Project Officer: Lasat, Mitch
Project Period: August 1, 2019 through July 31, 2022
Project Amount: $849,811
RFA: Advancing Actionable Alternatives to Vertebrate Animal Testing for Chemical Safety Assessment (2018) RFA Text |  Recipients Lists
Research Category: Safer Chemicals

Description:

Birth defects that affect musculoskeletal tissues account for 5% of all infant deaths. They may be caused by chemical ingredients in pesticides, fungicides, paints and food additives imposing substantial burdens on the affected individuals and families. Evaluating the safety of such chemicals using a suitable prenatal model of human embryos is therefore an essential scientific and societal goal. Currently, such evaluation is in vivo, mostly in rodents (integrated into OECD TG414, 415, 416, 421, 422), making it animal-, time- and cost-intensive. To spare animals, reduce cost and labor a variety of embryotoxicity assays have been proposed. However, these tests have mixed utility for routine industrial use, either because they still require the killing of animals or they have a relatively low predictivity for effects in humans. Consequently, innovative in vitro alternatives that adhere to the 3Rs while simultaneously being cost-effective and predictive of human risk are desperately needed.

Objective:

We hypothesize here that cultures of human embryonic stem cells (hESCs) can be utilized to predict the skeletal embryotoxicity of industrial and environmental chemicals in vitro. The objectives of this study are to osteogenically differentiate hESCs with concomitant chemical exposure (the test bank will include selected chemicals from the ToxCast I library with known effects on the skeleton). Detrimental effects of the chemical will be evaluated based on a reduction in matrix mineralization (as a surrogate of skeletal maturation).

Approach:

To identify suitable assays for matrix mineralization that adequately predict human risk, we will compare inexpensive absorbance-based assays and image analysis tools recently developed by us. Hierarchies of chemical potency and efficacy will be established by setting the half-maximal inhibitory doses of differentiation inhibition in relation to the half-maximal cytotoxic dose using a biostatistical model, canonical linear discriminants and integration of our new data into EPA’s Toxicology Priority Index to create a skeletal embryotoxicity score. A case study will determine whether the scoring system is sufficiently sensitive to distinguish between closely related chemical derivatives and will consequently establish whether the assay can be used to identify safer chemical alternatives. Lastly, testing the chemicals with differentiation protocols that generate only neural crest or mesodermal osteoblasts coupled with next-generation sequencing will identify potential modes-of-action (disruption of organogenesis) and mechanisms of toxicity (molecular-level initiating event).

Expected Results:

The proposed work is expected to achieve a classification of test chemicals by toxicologically relevant AOPs and based on an improved understanding of targeted effects on organogenesis, while at the same time achieving high predictivity at reduced labor and cost.

Supplemental Keywords:

chemicals, exposure, risk assessment, sensitive populations, dose-response, teratogen, biology, alternatives, human health