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Description of key information

The test material can tentatively be regarded a phenobarbitone-type inducer of liver cytochrome P450.

Additional information

Several studies were performed to assess the possible inducing effect of the test material on cytochrome P450 isoenzymes in the liver of rats. The first of which explored the possible inducing effect of the test material on cytochrome P450 isoenzymes, the subsequent studies investigated the dose-response relationship of the inducing effect of the test material, the latest study attempted to phenotype the liver induction effect of the test material. From the available data it can be concluded that the test material can tentatively be regarded a strong phenobarbitone-type inducer of liver cytochrome P450.

In a study to investigate the possible inducing effect of the test material on cytochrome P450 isoenzymes in rat liver, hepatic tissue samples were taken from control and high dose (1000 mg/kg) male rats, from a 28 -day oral gavage study, fixed in 4% buffered formalin and embedded in paraffin. Induction of cytochrome P450 isoenzymes in hepatocytes was then assessed by immunohistochemical staining of the liver sections with monoclonal antibodies, selected against a range of cytochrome P450 isoenzymes. Expression of rat liver cytochrome P450 was assessed semi quantitatively by comparison of staining intensity of sections from treated animals with those from (untreated) controls and positive controls.

Under the conditions of the study, isoenzymes of cytochrome P450 family CYP1A were found to be below the detection limit in all control and treated animals, isoenzyme of cytochrome P450 family CYP2B were found to be strongly induced by treatment with the test material with a centrilobular distribution pattern, isoenzyme of cytochrome P450 family CYP3A were found to be slightly induced by treatment with the test material with a centrilobular distribution pattern, and isoenzyme of cytochrome P450 family CYP4A were found to be slightly induced by treatment with the test material with a centrilobular distribution pattern

In conclusion, the test material can tentatively be regarded a strong phenobarbitone-type inducer of liver cytochromes P450.

 

In a study to investigate the dose-effect relationship of a possible inducing effect of the test material on cytochrome CYP2B isoenzymes in the liver of male and female rats, hepatic tissue samples were taken from five male, and five female, rats dosed with 0, 1000, 4000, 12000 or 20000 ppm test material in feed, of a 28 -day feeding study, fixed in 4% buffered formalin and embedded in paraffin. Induction of cytochrome CYP2B isoenzyme in hepatocytes was then assessed by immunohistochemical staining of the liver sections with the monoclonal antibody be4, selected against a range of cytochrome CYP2B1 and CYP2B2 isoenzymes. Expression of rat liver cytochromes P450 was assessed semi quantitatively by comparison of staining intensity of sections from treated animals with those from untreated (negative) and positive controls.

Under the conditions of the study, treatment with 0, 1000, 4000, 12000 or 20000 ppm test material resulted in a dose-related increase in staining intensity for both males and females, whereby the lowest dose resulted in a minimal induction of CYP2B isoenzymes, if any, and the highest dose resulted in a marked/strong (males) or moderate/marked (females) induction of CYP2B isoenzymes. Animals displaying minimal, slight or moderate levels of induction mostly presented with a centrilobular distribution pattern of CYP2B isoenzymes, whereas animals showing marked or strong enzyme inductions mostly presented with a panlobular distribution pattern.

 

In a study to investigate the dose-effect relationship of a possible inducing effect of the test material on cytochrome CYP2B isoenzymes in the liver of male and female rats, hepatic tissue samples were taken from four male, and four female, rats dosed with 0, 4000, 8000, or 16000 ppm test material in feed, of a 3 -month feeding study, fixed in 4% buffered formalin and embedded in paraffin. Induction of cytochrome CYP2B isoenzyme in hepatocytes was then assessed by immunohistochemical staining of the liver sections with the monoclonal antibody be4, selected against a range of cytochrome CYP2B1 and CYP2B2 isoenzymes. Expression of rat liver cytochromes P450 was assessed semi quantitatively by comparison of staining intensity of sections from treated animals with those from untreated (negative) and positive controls.

Under the conditions of the study, treatment with 0, 4000, 8000, or 16000 ppm test material resulted in a dose-related increase in staining intensity of CYP2B isoenzymes for both males and females. Animals displaying minimal to marked levels of expression mostly presented with a centrilobular distribution pattern of CYP2B isoenzymes, whereas animals showing strong enzyme expression presented with a panlobular distribution pattern.

 

In a study intended to phenotype biochemically and immunochemically a possible liver enzyme induction in male rats after administration of test material, groups of 5 male rats were treated for 14 consecutive days with dietary concentrations of 0, 1000, 2000, 5000 and 10000 ppm. One additional group of 5 rats was treated with phenobarbitone, a known potent model inducer of cytochrome P450 isoenzymes of subfamily CYP2B, for 14 consecutive days at a dietary concentration of 1000 ppm. In order to test for the reversibility of treatment-related changes, two groups of 5 male rats each were treated for 14 consecutive days with test material at dietary concentrations of 0 and 10000 ppm followed by a 28 -day recovery period.

During the in-life period, no mortality occurred and no clinical signs of toxicity were recorded. Treatment had no effect on bodyweight development or food consumption. At necropsy, no apparent macroscopic changes were observed although treatment at 5000 and 10000 ppm slightly increased relative liver weight. Effects on the liver microsomal cytochrome P450 system comprised: a slight to moderate induction of subfamily CYP2B isoenzyme at 5000 and 10000 ppm, as induced by increased activities of CYP2B-specific 7 -pentoxy and 7 -benzyloxyresorufin O-dealkylases as well as testosterone 16β-hydroxylase; a slight induction of subfamily CYP2A isoenzymes at 5000 and 10000 ppm, as indicated by increased activities of testosterone 6α-, 7α- and 15α-hydroxylases; and a minimal to slight induction of subfamily CYP3A isoenzymes at 10000 ppm, as indicated by increased activities of testosterone 6β- and 15β-hydroxylase as well as CYP3A1/2 protein contents.

Treatment was without an inducing effect on subfamily CYP1A isoenzymes and CYP1A2 protein content was only minimally induced. Likewise treatment was without an inducing effect on subfamily CYP4A isoenzymes. Effects on liver phase-II enzymes comprised moderately increased activity of microsomal epoxide hydrolase at 10000 ppm, as well as slightly to moderately increased activities of microsomal UDP-glucuronosyl-transferase and of cytosolic glutathione S-transferase at 5000 and 10000 ppm.

Treatment was without effect on cyanide insensitive peroxisomal fatty acid β-oxidation activity. Except for a slightly increased testosterone 6α-hydroxylase activity and, minimally increased CYP3A1/2 protein contents, all biochemical effects in the liver were fully reversible after a 28 day recovery period following treatment for 14 days at 10000 ppm.

Treatment with phenobarbitone at 1000 ppm slightly and moderately increased absolute and relative liver weights, respectively. As expressed, this model inducer had prominent effects on the liver microsomal cytochrome P450 system which comprised: a strong induction of subfamily CYP2B isoenzymes as indicated by increased CYP2B-specific activities of 7 -pentoxy and 7 -benzyloxyresorufin O-dealkylases as well as testosterone 16β-hydroxylase; a marked induction of subfamily CYP3A isoenzymes as demonstrated by increased CYP3A protein contents accompanied by increased activities of testosterone 2β-, 6β- and 15β-hydroxylases and a moderate induction of subfamily CYP2A isoenzymes as indicated by increased activities of testosterone 6α-, 7α- and 15α-hydroxylases.

Phenobarbitone was without any significant inducing effects on subfamily CYP1A isoenzymes and only minimally affected CYP1A-specific activities of 7 -methoxy and 7 -ethoxyresorufin O-dealkylases. Likewise treatment was without an inducing effect on subfamilyCYP4A isoenzymes. Effects on liver phase-II enzymes comprised moderately to markedly increased activities of microsomal epoxide hydrolase and microsomal UDP-glucuronosyl-transferase as well as of cytosolic glutathione S-transferase. Treatment was without an inducing effect on cyanide insensitive peroxisomal fatty acid β-oxidation activity.

Under the conditions of the study, the test material was identified as a slight to moderate liver enzyme inducer in the rat. Upon dietary administration for 14 days, the observed induction pattern showed similarities to that produced by the model inducer phenobarbitone. A mode of action of the test material as a polycyclic aromatic hydrocarbon- or peroxisome proliferator-type inducer can be excluded. The dose of 2000 ppm represents a no effect level in this study and the observed effects were essentially reversible upon a recovery period of 28 days.

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