of the risk. Carcinogenicity Musk xylene also demonstrates some of the problems of classifying substances as carcinogens. It has been placed into Group 3 ("not classifiable as to their carcinogenicity to humans") by the International Agency for Research on Cancer (IARC), and is classified in the European Union as category 3 carcinogen ("cause concern for man owing to possible carcinogenic effects but in respect of which the available information is not adequate for making a satisfactory assessment") under the Dangerous Substances Directive and a category 2 carcinogen ("suspected human carcinogen") under the CLP Regulation. These classifications are based mainly on a single study of oral exposure to musk xylene in B6C3F1-strain mice. The mice showed a highly significant increase in liver adenomas and carcinomas at median dietary intakes of 170 mg/kg body weight (males) and 192 mg/kg body weight (females), as well as significant increases in adenomas in the Harderian gland (male mice only) and in the liver at median dietary intakes of 91 mg/kg body weight (males) and 101 mg/kg body weight (females). The European Union Risk Assessment Report makes a number of observations about this study: it was conducted on a single species; no studies are available on, for example, rats; B6C3F1-strain mice are known to be particularly prone to liver cancers; the doses were high, and toxic effects (especially on the liver) were observed in the test animals; the mechanism of tumour development is unclear. Musk xylene is not genotoxic. It has significant effects on liver function which are similar to those shown by phenobarbital, for example, induction of CYP2B6 and other cytochrome P450 enzymes. The human carcinogenicity of phenobarbital has been the subject of debate, but it is currently classified in group 2B by the IARC and this appears to have been an important consideration in the classification of musk xylene as a category 3 carcinogen under the Dangerous Substances Directive. Nevertheless, the European Union Risk Assessment Report admits that musk xylene is a "borderline case". A further complication is the metabolism of musk xylene. One route of metabolism is through reduction of one or more nitro groups by the intestinal microflora (gut bacteria) to produce aromatic amines such as p-NH2-musk xylene. This metabolite has a different liver toxicity: in particular, it inhibits the CYP1B enzymes by covalent binding. Induction of cytochrome P450 enzymes, the most likely cause of rodent carcinogenicity, is a threshold phenomenon, with