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TRICLOSAN-INDUCED HEPATOCYTE PROLIFERATION
Special scientifically acceptable studies conducted in mice, rats, and hamsters have evaluated and compared the response of each species to triclosan with regard to hepatocellular proliferation:
Cell proliferation in rodent liver, mouse (Pathology Associates, Inc. 75N-0183H)
Triclosan and replicative DNA synthesis in hepatocytes, male rats (Ciba-Geigy Ltd CB 92/28-2)
Triclosan and replicative DNA synthesis in hepatocytes, hamster (RCC Project 356490.)
TRICLOSAN-INDUCED PEROXISOMES PROLIFERATION
Special studies conducted in mice, rats, and hamsters have evaluated and compared the response of each species to triclosan with regard to peroxisomes proliferation:
Hepatic biochemistry in liver in the mouse (Ciba-Geigy CB 91/18)
Hepatic biochemistry in liver in the rat (Ciba-Geigy CB 92/28)
Hepatic biochemistry in liver in the hamster (Ciba-Geigy CB 93/40)

Additional information

TRICLOSAN-INDUCED HEPATOCYTE PROLIFERATION

The effect of triclosan on hepatocyte replicative DNA synthesis has been examined in studies conducted in mouse, rat and Syrian hamster at doses ranging from 25 to 900 mg/kg bw/day (Pathology Associates, Inc. 75N-0183H; Ciba-Geigy Ltd CB 92/28-2; RCC Project 356490). The effect of triclosan on replicative DNA synthesis was monitored by using the proliferating cell nuclear antigen (PCNA) technique.

Immunohistochemical analyses of liver sections have shown increased numbers of S-phase cells in triclosan-treated mice, while histological examinations have revealed dose-dependent increases in the extent and severity of necrosis in mouse, but not in rat and hamster, liver.

The available data from special investigations in rodents demonstrate that in the mouse, treatment with triclosan for 90 days results in increased hepatocellular necrosis and hepatocyte proliferation starting at doses as low as 75 mg/kg bw/day in the diet.

In contrast, triclosan doses of up to 500 mg/kg bw/day (6000 ppm) in the rat did not result in increased cell proliferation. Evaluations in hamsters after 7 or 13 weeks of dosing at the level of 900 mg/kg bw/day in the diet showed no increases in cell proliferation compared to controls.

Based on evaluations of the cytotoxicity and necrosis observed in mouse livers, the data indicated that the hepatocyte proliferation was most likely a regenerative response secondary to cytotoxicity/necrosis resulting from liver damage induced by triclosan (i.e., the proliferation is compensatory). Altogether, the considerable data strongly show that, in clear contrast to the rat and hamster, triclosan increases cell proliferation in mouse liver, consistent with the liver being the sole target organ for tumour development in this species.

TRICLOSAN-INDUCED CHANGES IN LIVER PARAMETERS, PEROXISOMES PROLIFERATION

A series of three critical studies were conducted in mice, rats (males only), and hamsters to examine the effects of triclosan on selected biochemical and morphological liver parameters following dietary administration of triclosan for 14 days (Ciba-Geigy CB 91/18; Ciba-Geigy CB 92/28; Ciba-Geigy CB 93/40). Biochemical investigations included measurements of levels of protein and cytochrome P450 (CYP) content and CYP enzyme activities. Morphology was examined using electron microscopy (EM).

Dramatic differences were seen in responses between species. There was little difference between sexes, although females appeared slightly less sensitive to the effects of triclosan than males. Liver weight and microsomal CYP content were strongly increased in mice, mildly increased in higher dose rats and relatively unaffected in hamsters.

Similarly, induction of various enzyme activities was most pronounced and occurred at lower doses in mice when compared to other species.

Cyanide-insensitive fatty acid beta-oxidation activity (palmitoyl-CoA oxidation), a diagnostic indicator of peroxisome proliferation, showed a dose-dependent increase in mice, but not in rats or hamsters. Cytosolic glutathione S-transferase activity was mildly increased in mice, less so in rats, and slightly depressed in hamsters.

Lauric acid 12-hydroxylation activity, as catalysed by isoenzymes of the CYP4A gene subfamily, was strongly increased in mice but less so in rats and hamsters. Lauric acid 11-hydroxylation activity, as catalysed by isoenzymes of the CYP1A, CYP2B, and CYP2C families, was again strongly increased in mice, but less so in hamsters. No effects were found in rats.

Ethoxyresorufin O-deethylase (EROD) activity showed a mild dose-dependent increase in mice, with lesser increases in hamsters, and was decreased in rats. A strong induction of 7-pentoxyresorufin O-depentylase (PROD), a CYP2B marker, was found in mice and rats; hamsters showed significantly less potent induction of this activity.

The pattern of regio- and stereo-selective testosterone hydroxylation was used as a tool to assess treatment-related effects on the activities of several isoenzymes of the microsomal CYP enzyme family. Only in the mouse was total testosterone hydroxylation significantly induced. At the highest dose level of 951 mg/kg body weight/day, a 6-fold dose-dependent increase was observed. The most prominent changes in the testosterone hydroxylation profile were increased production of the 2 beta-, 6 beta-, 15 beta-, and 16 beta-hydroxy metabolites.

Production of the 2 beta- and 6 beta-hydroxy metabolites is associated with the expression of CYP3A, while 16 beta-hydroxylation is catalysed by the CYP2Be family. Rats showed a different pattern of testosterone hydroxylation after triclosan administration.

Production of the 16 beta-hydroxy metabolite was most prominent and, except for a mild induction of 15 alpha-hydroxylation which is associated with CYP2C12 and CYP2C13, no other hydroxy metabolite was produced in significant amounts. Hamsters were much less sensitive to CYP enzyme induction than mice. The only significant change observed in the hydroxylation profile was a small increase in 17-oxidation to form androstenedione.

Immunoblot analyses using monoclonal antibodies generated against, and specific for, inducible isoenzymes of rat liver cytochrome P450 were performed to further clarify the nature of triclosan-induced changes in liver enzymes. Mice demonstrated a slight dose-dependent decrease in the content of the isoenzymes of the CYP1A gene subfamily. Rats had an increased content of this isoenzyme family and hamsters a marginal increase. Triclosan strongly induced isoenzymes of the CYP3A subfamily in mice (8.4X control levels), produced a small increase (2X) at the highest dose in rats, and produced a decrease of this enzyme in female hamsters, with no effect in male hamsters. CYP4A induction was also extremely strong (7.8X at the highest dose) in mice and much less so in the rat and hamster (1.6X and 2.4X at the highest dose, respectively).

CONCLUSION

In assessing the data and interpreting the findings from all of repeated dose and carcinogenicity studies, it was important to evaluate the differences between the rodent species, specifically mice, rats, and hamsters. Liver biochemical, cell proliferation, and morphological responses to triclosan were investigated in a series of studies in all 3 rodent species. Triclosan showed peroxisome proliferator-type effects in the liver of mice (e.g., induction of large increases in peroxisomal fatty acid beta-oxidation, 11- and 12-hydroxylation of lauric acid, and levels of CYP4A proteins, together with increases in the numbers and size of peroxisomes), but not in rat or hamster livers at the doses tested.

It is notable that triclosan induced hepatic cell proliferation in the mouse, and not in the hamster or rat, in investigational studies of replicative DNA synthesis. Taking into account the results from these special investigations, sub-chronic toxicity data indicating an increased sensitivity in mice to triclosan’s hepatic effects, and pharmacokinetic data showing greater exposure levels to triclosan in mice compared to rats or hamsters, there is strong evidence that triclosan has peroxisome proliferator effects in mouse liver, but not in rat or hamster liver.

Given the association of peroxisome proliferation, cell proliferation, and tumour induction reported in the mouse, but no effects of these types in rats and hamsters, it was concluded that the mouse is uniquely sensitive to triclosan in the liver.

Importantly, it is generally accepted that chemicals which induce peroxisome proliferation and result in rodent hepatocarcinogenicity do not pose a health risk to humans. As such, the mouse is considered not to be a relevant or appropriate animal model for the evaluation of the tumourigenic potential of triclosan in humans.

As the hamster is the rodent species most comparable to humans based on pharmacokinetics (ADME) data and, therefore, the most appropriate model for the assessment of triclosan safety in humans, the lack of tumour development in this species provides the most conclusive non-clinical evidence for the absence of human carcinogenic potential.

This conclusion is supported strongly by the absence of effects of triclosan in a wide variety of in vitro and in vivo genotoxicity assays.