Screening and Evaluation of Male Reproductive Effects of Isothiazolinones at Human-Relevant Levels in Mammals: Direct Impairments and Increased Susceptibility to Subsequent Spermatogenic Stress

Although isothiazolinones (ITs) are widely used as antimicrobials in various products, the understanding of their hazards remains incomplete. This study aimed to evaluate the adverse effects of ITs at human-relevant levels on the male mammal reproductive system. First, we employed testicular cells to determine all test ITs exerting relatively high toxicity, with 2-octyl-4-isothiazolin-3-one (OIT) exhibiting toxicity higher than that of other ITs. Then, the OIT was administered to mice through drinking water at nominal doses of 50 and 5000 μg/kg/day from gestational day 10. Consequently, we found that the two doses of the OIT caused retarded testis development in suckling pups, accompanied by male reproductive impairments in adulthood, including decreased sperm count and increased malformations. When receiving subsequent spermatogenic stress induced by an injection of busulfan, OIT-exposed mice exhibited more pronounced testicular abnormalities and sperm defects, indicating increased susceptibility to stress. In addition, our in vitro results of germ cell line and mouse sperm, combined with sperm defects in animals, suggest the possibility that OIT-caused testicular impairments are partly due to its binding to thiol-containing molecules. Altogether, our study for the first time shows that exposure to OIT at human-relevant levels not only impairs the male reproductive system but also induces it to be more susceptible to subsequent spermatogenic stress, which highlights reproductive health risks of OIT.