damage and compensatory cell proliferation. Support for this mechanism comes from studies of oxidative DNA damage induced by o-toluidine metabolites in cultured human cells (HL-60), calf thymus DNA, and DNA fragments from key genes thought to be involved in carcinogenesis (the c-Ha-ras oncogene and the p53 tumor-suppressor gene). Also supporting this mechanism are observations of o-toluidine-induced DNA damage (strand breaks) in cultured human bladder cells and bladder cells from rats and mice exposed in vivo to o-toluidine. Binding of hemoglobin Metabolites of o-toluidine bind hemoglobin in rats. The relevant metabolite is thought to be o-nitrosotoluene. which also causes urinary-bladder cancer in rats. Nitrosotoluene converts hemoglobin to methemoglobin, resulting in methemoglobinemia. Evidence suggests that this pathway is relevant to humans. Carcinogenicity Although the mechanisms of carcinogenicity of o-toluidine are not completely understood, the available evidence suggests that they are complex and involve several key modes of action, including metabolic activation that results in binding of reactive metabolites to DNA and proteins, mutagenicity, oxidative DNA damage, chromosomal damage, and cytotoxicity. In the U.S., o-toluidine was first listed in the Third Annual Report on Carcinogens as ‘reasonably anticipated to be a human carcinogen’ in 1983, based on sufficient evidence from studies in experimental animals. The Report on Carcinogens (RoC) is a U.S. congressionally-mandated, science-based public health report that identifies agents, substances, mixtures, or exposures in the environment that pose a hazard to people residing in the United States Since then, other cancer related studies have been published and the listing of o-toluidine was changed to ‘known to be a human carcinogen’. o-toluidine was especially linked to bladder cancer. This was done 31 years later in the Thirteenth Report on Carcinogens (2014). The International Agency for Research on Cancer (IARC) has classified o-toluidine as ‘carcinogenic to humans (group 1)’. Toxicology The main excretion-pathway is revealed to be through urine where up to one-third of the administered compound was recovered unchanged. o-toluidine and metabolites are known to bind to hemoglobin. The o-toluidine metabolite o-nitrosotoluene, is proven to cause bladder cancer in rats and is thought to bind to hemoglobin in humans. o-Toluidine exposure has been researched in a number of different degrees in animals. Single exposure o-Toluidine was found to be harmful to rats following acute oral exposure with LD50 of 900 and 940 mg/kg bodyweight. The compound was also found to be of low toxicity in rabbits following acute dermal exposure with an LD50