Bioactivity of the ubiquitous tire preservative 6PPD and degradant, 6PPD-quinone in fish- and mammalian-based assays

Abstract

6PPD-quinone (N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone), a transformation product of the antiozonant 6PPD (N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine) is a likely causative agent of coho salmon (Oncorhynchus kisutch) pre-spawn mortality. Stormwater runoff transports 6PPD-quinone into freshwater streams, rapidly leading to neurobehavioral, respiratory distress, and rapid mortality in laboratory-exposed coho salmon, but causing no mortality in many laboratory-tested species. Given this identified hazard, and potential for environmental exposure, we evaluated a set of U.S. Environmental Protection Agency's high-throughput assays for their capability to detect the large potency difference between 6PPD and 6PPD-quinone observed in coho salmon and screen for bioactivities of concern. Assays included transcriptomics in larval fathead minnow (FHM), developmental and behavioral toxicity in larval zebrafish, phenotypic profiling in a rainbow trout gill cell line, acute and developmental neurotoxicity in mammalian cells, and reporter transcription factor activity in HepG2 cells. 6PPD was more consistently bioactive across assays, with distinct activity in the developmental neurotoxicity assay (mean 50th centile activity concentration = 0.91 µM). Although 6PPD-quinone was less potent in FHM and zebrafish, and displayed minimal neurotoxic activity in mammalian cells, it was highly potent in altering organelle morphology in RTgill-W1 cells (phenotype-altering concentration = 0.024 µM compared with 0.96 µM for 6PPD). Although in vitro sensitivity of RTgill-W1 cells may not be as sensitive as intact Coho salmon, the assay may be a promising approach to test chemicals for 6PPD-quinone-like activities. The other assays each identified unique bioactivities of 6PPD, with neurobehavioral and developmental neurotoxicity being most affected, indicating a need for further assessment of this chemical. Our results demonstrate that the common tire additive, 6PPD, is bioactive in a broader set of assays than the environmental transformation product 6PPD-quinone and that it may be a developmental neurotoxicant in mammals, whereas 6PPD-quinone was much more potent than 6PPD in altering the intracellular phenotype of rainbow trout gill cells. Application of the set of high-throughput and high-content bioassays to test the bioactivity of this emerging pollutant has provided data to inform both ecological and human health assessments.

Keywords: cell painting; high-throughput screening; neurotoxicity; storm water contaminants.