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.