Implementing Toxicity Testing in the 21st Century (TT21C): Making safety decisions using toxicity pathways, and...

Area of Science:

• Toxicology
• Risk Assessment
• In Vitro Methods

Background:

• Traditional toxicology relies on high-dose animal studies and extrapolation for risk assessment.
• The Toxicity Testing in the 21st Century (TT21C) initiative proposes a shift towards in vitro methods using human-relevant cells.
• Adverse Outcome Pathways (AOPs) expand on this concept, but practical implementation remains a challenge.

Purpose of the Study:

• To provide a practical example of implementing the TT21C vision for chemical safety assessment without animal testing.
• To develop and test a strategy for a pathway-based risk assessment using a case study approach.
• To explore the feasibility of constructing a TT21C risk assessment using in vitro dose-response data for a specific chemical.

Main Methods:

• Utilized a prototype toxicity pathway (DNA damage responses mediated by the p53 network).
• Conducted a case study using quercetin, a chemical with defined human exposure.
• Generated high-throughput pathway biomarker data and cell-cycle/apoptosis markers in HT1080 cells using flow cytometry and imaging.
• Performed biokinetic modeling and explored in vitro to in vivo extrapolation.

Main Results:

• Generated 18-point dose-response curves to identify concentrations causing significant cellular perturbation.
• Compared No Observed Effect Levels (NOELs) and Benchmark Doses (BMDs) with biokinetic modeling outputs.
• Conducted a first-tier risk assessment by comparing in vitro and predicted in vivo quercetin concentrations.
• Identified shortcomings and areas for improvement in the pathway-based risk assessment process.

Conclusions:

• The study presents a progress report on an ongoing effort to establish a proof-of-concept in vitro-only safety assessment.
• Demonstrates the potential of pathway-based approaches for chemical safety evaluation, reducing reliance on animal testing.
• Highlights the need for further development to fully implement TT21C principles into practical risk assessment tools.