Hazardous effects of 6PPD on human small intestinal organoids

N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD) is the most ubiquitously utilized antioxidant in the manufacture of rubber products. While it has been associated with the development of human colorectal cancer (CRC), its intestinal hazardousness in humans remains experimentally unconfirmed. In this study, human small intestinal organoids were used to assess the intestinal toxicity of 6PPD. The findings of this study demonstrated that exposure to 6PPD at concentrations ranging from 300 to 900 μg·L⁻¹ resulted in impaired organoid growth in a concentration-dependent manner, as evidenced by reduced organoid count and size. Although even the highest concentration tested (900 μg·L⁻¹) did not induce overt cell death, viability was significantly inhibited across this concentration range, as evidenced by decreased Calcein fluorescence and CCK-8 metabolic activity. Immunofluorescence analysis further revealed suppressed proliferation and downregulation of epithelial and goblet cell markers, whereas markers of Paneth, villus, and enteroendocrine cells remained unchanged. Notably, at higher concentrations (≥1000 μg·L^−1^), 6PPD rapidly induced organoid disintegration and significant increases in cell death. Together, these results demonstrate that 6PPD disrupts human intestinal epithelial development and survival in a concentration-dependent manner, providing direct experimental evidence of its cytotoxic potential in human guts and highlighting the need for further mechanistic and regulatory investigations.