ACTIVATION BY CECAL REDUCTION OF THE AZO-DYE-D-AND-C-RED-NO-9 TO A BACTERIAL MUTAGEN

D and C Red No. 9 is a monoazo dye used for manufacturing printing inks, rubber and plastics, and as an additive in cosmetics and drugs. In an NTP carcinogenicity study in rats and mice it induced splenic sarcomas and liver nodules in male rats; no chemical-related tumours were induced in mice. On the basis of its contradictory responses in a range of in vitro tests and its inactivity in several bl vivo genotoxicity assays, it has been suggested that the dye may act as a non-genotoxic carcinogen. We tested the dye in the Salmonella mutagenicity assay using several different protocols. The dye was not mutagenic when tested using the standard (aerobic) preincubation protocol. Variable responses were seen when the flavin mononucleotide (FMN) reduction protocol was used. A third protocol was provided by incubating the test compound overnight with a rat caecal preparation under anoxic conditions to reduce the azo bond. Ethyl acetate extracts of this incubation mixture, when tested in the standard preincubation protocol using induced rat liver S9, yielded dose-related mutagenic responses in TA100, and a weak response in TA98. The presumed major reduction product, 1-amino-2-naphthol (1-A-2-N) was mutagenic to TA100, but not TA98, in standard protocols with S9. The results show that it is necessary to use a protocol in which D and C Red No. 9 is reduced in order to demonstrate the mutagenicity of this dye. The non-genotoxicity previously reported for D and C Red No. 9, may have been due to insufficient reductive cleavage. The carcinogenicity of this compound may, therefore, be a consequence of its genotoxicity, rather than a result of some non-genotoxic process.