Evaluating Bioactivity of Tire Preservative, 6PPD, and its Degradant, 6PPD-quinone, in High Throughput Assays
For a general overview of 6PPD-quinone and EPA's actions to address it, visit the 6PPD-quinone webpage.
6PPD (N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine) is a chemical added to most automobile tires to protect rubber from reactions with oxygen and ozone that can reduce tire life-time due to cracking and degradation. When 6PPD reacts with atmospheric ozone, a transformation product, 6PPD-quinone, is formed that can leach from tires and tire wear particles and runoff to streams during storm events. In 2021, Tian et. al identified 6PPD-quinone as the probable cause of urban runoff associated pre-spawn mortality in coho salmon. Subsequent studies have confirmed the sensitivity of coho salmon and other salmonids to 6PPD-quinone, while many other fish species, including those commonly employed in toxicity testing, were found to be relatively insensitive.
Science Challenge
- Toxicity testing with sensitive salmonids is costly, time-consuming, and animal intensive.
- To assess possible alternatives (replacement chemicals) and/or management options such as using green infrastructure to intercept 6PPD-quinone in the field, there is a need to identify alternative toxicity testing methods (bioassays) that can detect 6PPD-quinone’s biological activity and are lower cost and higher throughput than toxicity testing with salmonids.
Assay Evaluation
- EPA evaluated its portfolio of rapid toxicity tests (i.e., New Approach Methodologies – NAMs) for methods that could detect the large potency difference between 6PPD and 6PPD-quinone that exists for sensitive salmonids.
- Three fish-based assays and three mammalian assays were evaluated.
| Assay Type | Model Organism |
|---|---|
| Global gene expression in larval fish | Fathead minnow |
| Developmental and behavioral toxicity in larval fish | Zebrafish |
| High-throughput phenotypic profiling with the Cell Painting assay | Rainbow trout gill cell line (RTgill-W1) |
| Acute neurotoxicity using micro-electrode arrays | Mammalian Cells |
| Developmental neurotoxicity battery (including neurite proliferation, outgrowth, synapse formation, neural network formation) | Mammalian Cells |
| Activation of nuclear receptors or G protein-coupled receptors | Mammalian Cells |
Results
- 6PPD elicited some response in all of the bioassays, but at relatively high concentrations.
- 6PPD-quinone was only more potent than 6PPD in the Cell Painting assay employing rainbow trout gill cells.
Primary Conclusion
- A phenotypic profiling (Cell Painting) assay using the RTgill-W1 cell line may be useful as a high-throughput screening assay for toxicity evaluation of environmental samples as well as potential 6PPD replacement chemicals.
Next Steps
- EPA will apply the RTgill-W1 Cell Painting assay to screen environmental samples with measured 6PPD and 6PPD-quinone concentrations.
- EPA will procure potential replacements for 6PPD and their environmental transformation products for evaluation in the RTgill-W1 Cell Painting assay.
- EPA will compare the RTgill-W1 Cell Painting assay to that of a coho salmon primary cell toxicity assay to evaluate relative sensitivity and costs.
More Information
Mark Jankowski, US EPA, Region 10 (Jankowski.Mark@epa.gov)
Joshua Harrill, US EPA, Center for Computational Toxicology and Exposure (harrill.joshua@epa.gov)