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(a)
Discussion of a possible mode of carcinogenic action of difenoconazole.
A. Postulated mode of action.
In mice and rats, treatment with difenoconazole caused an increasee in the incidences of hepatocellular
adenomas and carcinomas in male and female mice given diets containing difenoconazole at
2500 ppm, equal to 423 and 513 mg/kg bw per day, respectively, for 18 months (Cox et al., 1989a), but
not in male or female rats given the same diet, equal to 124 and 170 mg/kg bw per day, respectively, for
24 months (Cox et al., 1989b). Difenoconazole has also been shown to induce various hepatic enzymes
of xenobiotic metabolism and it is not genotoxic. These general properties suggest that difenoconazole
could have a mode of carcinogenic action (MOA) similar to that of phenobarbital. The generally
proposed MOA for phenobarbital is tumour promotion resulting from a long-standing hepatomegaly,
hepatocellular hypertrophy and hyperplasia of the liver. Treatment with phenobarbital alone produces
hepatocellular adenomas and carcinomas in mice and, when administered after a short treatment with
a known liver carcinogen, the incidences of tumours are increased. The potency of CYP induction in
mice and rats correlates with the degree of tumour promotion. In addition, phenobarbital has mitogenic
effects in liver and has been shown in many studies to inhibit intercellular communication (reviewed
in IARC, 2001). Phenobarbital has been studied to a far greater degree than difenoconazole and so
it is not expected that there should be complete concordance of the data in order to conclude that the
MOAs are most likely the same; however, clear discord should not be demonstrated.
B. Key events.
Difenoconazole is expected to induce hepatic CYP enzymes. A study on such an effect was
performed in male TIF:Magf (SPF) mice given difenoconazole at a dose of 0, 1, 10, 100 or 400 mg/kg
bw per day for 14 days. Reversibility was studied in the same experiment in which groups were given
difenoconazole at 0 or 400 mg/kg bw per day for 14 days followed by 28 days without treatment. Other
groups were given phenobarbital, 3-methylcholanthrene or nafenopin (Thomas, 1992). Difenoconazole
increased liver weight, hepatic cytochrome P450 protein and a number of hepatic CYP enzyme
and other microsomal (UDP-glucuronosyltranferase, lauric acid 11- and 12-hydroxylase, epoxide
hydrolase) enzyme activities, as well as microsomal cytochrome P450-dependent testosterone hydroxylation
in a way that was generally very similar to that observed in the same strain of male mouse
when phenobarbital was administered. Most effects were observed in only the groups at 100 and 400
mg/kg bw per day, although certain enzyme activities were increased even with difenoconazole at
1 mg/kg bw per day. All effects were dose-dependent and regressed within the 28-day recovery period.
Usually, regression was complete. The pattern of enzyme induction by difenoconazole was very
similar to that of phenobarbital for 7-ethoxyresorufin O-deethylase (EROD) and 7-pentoxyresorufin
O-depentylase (PROD) activities in particular. This similarity is important because it is quite different
from the induction pattern observed after treatment with either 2-methylcholanthrene or nafenopin.
Clearly, other key events must follow, but there was no experimental evidence with d ifenoconazole
to support their participation in an MOA.
C. Dose–response relationships.
All effects on enzyme induction in the 14-day study and the tumour response in the 18-month
study were dose-dependent. Liver-weight increases were dose-dependent in a 3-month study in mice. At
daily doses of 200 and 2500 ppm, equal to 34 and 440 mg/kg bw in males and 45 and 639 mg/kg bw in
females, the absolute liver-weight increases were 9% and 82% in males and 11% and 71% in females. In
rats, on the other hand, the responses were less clear. At daily doses of 750, 1500 and 3000 ppm equal to
51, 105 and 214 mg/kg bw in males and 66, 131 and 275 mg/kg bw in females, the absolute liver‐weight
increases were 25.7%, 30.9% and 30.2% in males and 24.5%, 26.2% and 40.2% in females.
DIFENOCONAZOLE 201–272 JMPR 2007