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Toxicological information

Additional toxicological data

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Administrative data

Endpoint:
additional toxicological information
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Performed under GLP conditions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Type of study / information:
- Type of effects studied: biochemical or cellular interactions (Porphyrinogenic potential & effects on selected liver parameters)
- in-vivo
Test guideline
Qualifier:
no guideline required
Principles of method if other than guideline:
Investigation of the porphyrinogenic potential and effects on selected liver parameters following dietary administration to the rat (male/female). For that purpose, groups of Sprague Dawley test organism (male/female) each were treated at dietary concentrations of 0, 300 and 4000 ppm for 14 consecutive days. At the end of the treatment period, the following investigations were performed:
- Histopathology of the liver.
- Determination of plasma amino transferases ALAT and ASA T.
- Determination of selected liver xenobiotic-metabolizing enzymes.
- Determination of bilirubin in plasma.
- Determination of porphyrin contents in liver, spleen, plasma, urine and faeces.
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(allyloxy)-2-methyl-1-oxopropan-2-yl 2-chloro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1(2H)-yl]benzoate
EC Number:
603-837-5
Cas Number:
134605-64-4
Molecular formula:
C20H18ClF3N2O6
IUPAC Name:
1-(allyloxy)-2-methyl-1-oxopropan-2-yl 2-chloro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1(2H)-yl]benzoate
Details on test material:
- Stability under test conditions: Stable under test conditions
- Storage condition of test material: Room temperature

Results and discussion

Any other information on results incl. tables

Details on results

IN-LIFE PHASE

Mean daily food consumption ratio (MFC) and mean daily dose:

At 4000 ppm, mean daily food consumption ratio was markedly lower at study day 2 in both sexes. MFC at both dose levels was in the range of the control group, when averaged over the whole treatment period. Calculated from the actual test article concentration in the diet, the mean daily doses for males/females were 21.3/21.9 and 258.3/269.3 mg/kg bw for the nominal concentrations of 300 and 4000 ppm in the diet, respectively.

 

Body weight development:

In males and females, treatment at 4000 ppm resulted in a slightly retarded body weight gain during the first 2 days of treatment. Thereafter, body weight gain was comparable to controls.

 

Mortality and clinical observations:

No mortality occurred throughout the in-life period and no clinical s1gns of toxicity were observed.

 

NECROPSY

Macroscopical findings:

All animals were sacrificed scheduled at study day 15. No apparent macroscopic changes were observed in males and females at necropsy.

Carcass weight:

At sacrifice, the carcass weight of all animals was recorded after bleeding. Treatment had no effect on carcass weight.

Absolute and relative liver weights:

In males, treatment at 4000 ppm moderately increased mean relative liver weight to 124% of control. In females, treatment had no effect on mean absolute and relative liver weights.

Absolute and relative spleen weights:

Treatment had no effect on mean absolute and relative spleen weights.

Absolute and relative adrenal weights:

Treatment had no effect on mean absolute and relative adrenal weights.

 

LABORATORY INVESTIGATIONS

Histopathology:

Upon microscopic examination, a hepatocellular hypertrophy was observed in males treated at 4000 ppm. Except for hepatocytes adjacent to central veins, which showed some accumulation of smooth endoplasmic reticulum, the hepatocellular enlargement was characterized by an increased, clear appearing cytoplasm, probably due to excess storing of glycogen. Among the top dose females, only one animal showed a similar change, although of lower severity. In addition, a slightly increased mitotic activity of hepatocytes was observed in males and females at 4000 ppm. Some other histopathologic findings were found in this study (apoptosis, inflammatory cell infiltration, single cell necrosis), which gave no indication of a treatment-related association with respect to their incidence, distribution or morphologic appearance.

 

Plasma parameters:

Aspartate aminotransferase (ASAT) activity:

Plasma ASAT acttvlty was determined as clinical parameter of liver damage and loss of hepatocellular membrane integrity. ASAT is mainly located in the mitochondria of hepatocytes. Treatment had no effect on plasma ASAT activity.

 

Alanine aminotransferase (ALAT) activity:

Plasma ALAT activity was determined as clinical parameter of liver damage and loss of hepatocellular membrane integrity. ALAT is mainly located in the cytosol of hepatocytes and is more specific for the liver than ASAT. In males, treatment at 4000 ppm minimally increased plasma ALAT activity to 222% of control. In females, treatment at 4000 ppm minimally increased plasma ALAT activity to 118% of control.

 

Bilirubin:

Plasma bilirubin was determined as clinical parameter for altered heme degradation. In males, treatment had no effect on plasma bilirubin concentration. In females, treatment at 4000 ppm moderately increased plasma bilirubin concentration to 159% of control.

 

Porphyrin contents:

The porphyrin content was determined in liver, spleen, plasma, urine and feces as a parameter for effects on porphyrin biosynthesis.

 

Liver:

In males, treatment at 300 and 4000 ppm moderately and strongly increased the hepatic porphyrin content to 200% and 700% of control, respectively. In females, treatment at 300 and 4000 ppm moderately and strongly increased the hepatic porphyrin content to 200% and 492% of control, respectivel.

 

Spleen:

In animals of both sexes, treatment at 4000 ppm moderately increased the porphyrin content in spleen to 287% and 220% of control, respectively.

 

Plasma:

In males. treatment at 300 and 4000 ppm moderately and strongly increased the plasma porphyrin content to 198% and 620% of control, respectively. In females, treatment at 300 and 4000 ppm moderately and strongly increased the plasma porphyrin content to 211% and 815% of control, respectively.

 

Urine:

In males, treatment at 300 and 4000 ppm strongly increased the urinary porphyrin content (nmol/ml urine) to 553% and 468% of control, respectively. The total amount of porphyrin excreted via urine during the last 24 h before sacrifice (nmol/24 h urine) was strongly increased to 617% of control at 4000 ppm. In females, treatment at 300 and 4000 ppm moderately and strongly increased the urinary porphyrin content (nmol/ml urine) to 375% and 1588% of control, respectively. Likewise, the total amount of porphyrin excreted via during the last 24 h before sacrifice (nmol/24 h urine) was moderately and strongly increased to 336% and 1778% of control at 300 and 4000 ppm, respectively.

 

Feces:

In males, treatment at 300 and 4000 ppm strongly increased the fecal porphyrin content (nmol/g wet feces) to 1184% and 1710% of control, respectively. Likewise, the total amount of porphyrin excreted in feces during the last 24 h before sacrifice (nmol/24 h feces) was strongly increased to 990% and 1819% of control at 300 and 4000 ppm, respectively. In females, treatment at 300 and 4000 ppm strongly increased the fecal porphyrin content (nmol/g wet feces) to 854% and 816o/c of control, respectively. Likewise, the total amount of porphyrin excreted in feces during the last 24 h before sacrifice (nmol/24 h feces) was strongly increased to 848% and 1033% of control at 300 and 4000 ppm, respectively.

 

Biochemical liver parameters:

Protein content of 100xg supernatant:

In males and females, treatment at 4000 ppm minimally decreased protein content of liver 100xg supernatant to 93% and 92% of control, respectively.

Protein content of microsomal fraction:

ln males, treatment at 4000 ppm slightly increased liver microsomal protein content to 123% of control. In females, treatment had no effect on liver microsomal protein content.

Protein content of cytosolic fraction: In males and females, treatment at 4000 ppm minimally decreased liver cytosolic protein content to 88% and 90% of control, respectively.

 

Microsomal cytochrome P450 content:

In males and females, treatment 4000 ppm slightly decreased liver microsomal cytochrome P450 content to 64% and 63% of control, respectively.

 

Microsomal 7-methoxy 0-demethylase (MROD):

In the rat liver, 0-demethylation of methoxyresorufin is most efficiently catalyzed by

cytochrome P450 isoenzyme CYP 1 A2]. The activity is strongly inducible by polycyclic aromatic hydrocarbons. In males, treatment at 4000 ppm moderately decreased liver microsomal MROD activity to 45% of control. In females, treatment at 300 and 4000 ppm slightly

decreased liver microsomal MROD activity to 68% and 66% of control, respectively.

 

Microsomal 7-ethoxy 0-deethylase (EROD):

In the rat liver, 0-deethylation of ethoxyresorufin is most efficiently catalyzed by cytochrome P450 isoenzymes CYP1A1 and CYP1A2. The activity is strongly inducible by polycyclic aromatic hydrocarbons. In males, treatment at 4000 ppm markedly decreased liver microsomal EROD activity to 29% of control. In females, treatment at 300 and 4000 ppm slightly and moderately decreased liver microsomal EROD activity to 68% and 51% of control, respectively.

 

Microsomal 7-pentoxy 0-depentylase (PROD):

In the rat liver, 0-depentylation of pentoxyresorufin is most efficiently catalyzed by cytochrome P450 isoenzyme CY2B1. The activity is strongly inducible by barbituric acid derivatives. In males, treatment at 4000 ppm strongly decreased liver microsomal PROD activity to 17% of control. In females, treatment at 300 and 4000 ppm slightly and markedly decreased liver microsomal PROD activity to 69% and 31% of control, respectively.

 

Microsomal 7-benzyloxy 0-debenzylase (BROD):

In the rat liver, 0-debenzylation of benzyloxyresorufin is catalyzed by cytochromes P450 isoenzymes of subfamily CYP2B and a variety of other cytochrome P450 isoenzymes. MROD is strongly inducible by barbituric acid derivatives. In males and females, treatment at 4000 ppm moderately decreased liver microsomal BROD activity to 48% and 53% of control, respectively.

 

Microsomal UDP-glucuronosyl transferase (UDPGT):

UDP-glucuronosyltransferases constitute a gene superfamily of enzymes exhibiting overlapping substrate specificity. In the rat, prototype enzyme inducers like 3-methylcholanthrene, phenobarbital, pregnenolone-16α-carbonitrile or peroxisome proliferators have different inducing effects on the isoenzymes of this gene superfamily. For the present study, liver microsomal UDPGT activity was determined using 3-methyl-2-nitrophenol. Treatment had no effect on liver microsomal UDPGT activity.

 

Peroxisomal β-oxidation (BOX):

β-0xidation of long chain fatty acids and fatty acid derivatives is one of the major physiological function of peroxisomes and represents a diagnostic parameter for the assessment of peroxisome proliferation in the rodent liver. In the rat liver, a strong induction of peroxisomal β-oxidation is observed after treatment of rats with peroxisome proliferators. In males, treatment at 4000 ppm moderately induced liver peroxisomal BOX activity to 236% of control. In females, treatment at 4000 ppm minimally induced liver peroxisomal BOX activity to 123% of control.

 

Immunoblot analyses of microsomal cytochrome P450 contents:

MAb d15 is a monoclonal antibody raised against the purified rat liver cytochrome P450 isoenzyme CYP1A2 and crossreacts with purified cytochrome P450 isoenzyme CYP1A 1. Isoenzymes of family CYP1A are known to be inducible by polycyclic aromatic hydrocarbons. In liver microsomes of untreated male rats, this antibody recognized one protein band with a molecular weight corresponding to cytochrome P450 isoenzyme CYP1A2. Treatment at 300 and 4000 ppm moderately decreased the intensity of this constitutive protein band to 58% and 47% of control. In liver microsomes of untreated female rats, this antibody recognized one protein band with a molecular weight corresponding to the constitutive protein band observed in males (CYP1A2). Treatment at 300 and 4000 ppm slightly decreased the intensity of

this constitutive protein band to 79% and 73% of control. This result indicates a treatment related decreased content of microsomal CYP1A2 and is in accordance with the slight to marked decrease of MROD and EROD activities in both sexes at 4000 ppm.

 

MAb be4 is a monoclonal antibody raised against and specific for rat liver cytochrome P450 isoenzymes CYP2B1 and CYP2B2, which are known to be strongly inducible by barbituric acid derivatives. In addition, this antibody recognizes a third, unknown cytochrome P450 isoenzyme, which is constitutively expressed in the rat 1iver and not inducible by phenobarbital. In liver microsomes of untreated male rats, this antibody recognized one protein band with a molecular weight corresponding to the third, constitutively expressed cytochrome P450 isoenzyme.

Treatment at 300 and 4000 ppm slightly and moderately decreased the intensity of this protein band to 72% and 54% of control, respectively. In liver microsomes of untreated female rats, this antibody recognized the same protein band as in males. Treatment at 300 and 4000 ppm slightly and moderately decreased the intensity of this protein band to 63% and 41% of control, respectively.

 

MAb p6 is a monoclonal antibody raised against and specific for rat liver cytochromes of gene family CYP3A, which are known to be inducible by steroids and phenobarbital. In liver microsomes of untreated male rats, this antibody recognized two protein bands corresponding to cytochrome P450 isoenzyme CYP3A1 (higher molecular weight) and the male specific cytochrome P450 CYP3A2 (lower molecular weight).

Treatment at 4000 ppm minimally increased the intensity of the CYP3A1 band to 124% of control and moderately decreased the intensity of the CYP3A2 band to 46% of control. In liver microsomes of untreated female rats, this antibody recognized one weak protein band with a molecular weight corresponding to the constitutively expressed CYP3A1 protein band as observed in males. Treatment at 4000 ppm markedly increased the intensity of this protein band to 257% of control.

 

MAb clo4 is a monoclonal antibody raised against and specific for rat liver isoenzymes of the cytochrome P450 gene family CYP4A, which are known to be inducible by peroxisome proliferators. In liver microsomes of untreated male rats, this antibody recognized two constitutive protein bands with molecular weights corresponding to cytochrome P450 isoenzymes CYP4A1/3 (higher molecular weight) and the male specific cytochrome P450 isoenzyme CYP4A2 (lower molecular weight). Treatment at 300 and 4000 ppm slightly increased the intensities of these protein bands to 168%/150% and 236%/207% of control, respectively. In liver microsomes of untreated female rats, this antibody recognized one constitutive protein band with a molecular weight corresponding to cytochrome P450 isoenzymes CYP4A1/3. Treatment at 4000 ppm slightly decreased the intensity of this protein band to 77% of control.

Applicant's summary and conclusion

Conclusions:
Upon subchronic dietary administration to rats, the test substance interfered with heme biosynthesis with the consequence of dose-dependent, pronounced porphyria and a decreased activity of various isoenzymes of the hepatic microsomal cytochrome P450 system. A weak potency of the test article to act as a liver peroxisome proliferator was observed in high dosed males.
Executive summary:

Groups of 5 young adult male and female Sprague Dawley rats were treated for 14 days with the test substance at dietary concentrations of 0, 300 and 4000 ppm, corresponding to mean daily doses of 0, 21.3 and 258.3 mg/kg body weight for males and of 0, 21.9 and 269.3 mg/kg body weight for females, respectively.

The test substance caused neither clinical signs of toxicity nor mortality. Body weight gain was slightly retarded at 4000 ppm in both sexes during the first two days and was thereafter comparable to controls. Likewise, mean daily food consumption ratio was reduced at study day 2 and was thereafter comparable to controls. Treatment had no effect on carcass weight in both sexes. The following effects were observed after the 14-day treatment period:

Effects observed at 4000 ppm (males: m; females: f):

-        relative liver weight was increased to 124% (m) of control

-        hepatocellular hypertrophy (m) was observed

-        mitotic activity of hepatocytes was increased (m,f)

-        plasma alanine aminotransferase activity was increased to 222% (m) and 118% (f) of control

-        plasma bilirubin content was increased to 159% (f) of control

-        liver porphyrin content was increased to 700% (m) and 492% (f) of control

-        spleen porphyrin content was increased to 287% (m) and 220% (f) of control

-        plasma porphyrin content was increased to 620% (m) and 815% (f) of control

-        urinary porphyrin content (nmol/ml urine) was increased to 468% (m) and 1588% (f) of control. Total urinary amount of porphyrin (nmol/24 h urine) was increased to 617% (m) and 1778% (f) of control

-        fecal porphyrin content (nmo1/g wet feces) was increased 1710% (m) and 816% (f) of control. Total fecal amount of porphyrin (nmol/24 h feces) was increased to 1819% (m) and 1033% (f) of control

-        liver protein content of 100xg supernatant was decreased to 93% (m) and 92% (f) of control

-        liver microsomal protein content was increased to 123% (m) of control

-        liver cytosolic protein content was decreased to 88% (m) and 90% (f) of control

-        liver microsomal cytochrome P450 content was decreased to 64% (m) and 63% (f) of control

-        liver microsomal 7-methoxyresorufin 0-demethylase activity was decreased to 45% (m) and 66% (f) of control

-        liver microsomal 7-ethoxyresorufin 0-deethylase activity was decreased to 29% (m) and 51% (f) of control

-        liver microsomal 7-pentoxyresorufin 0-depentylase activity was decreased to 17% (m) and 31% (f) of control

-        liver microsomal 7-benzyloxyresorufin 0-debenzylase activity was decreased to 48% (m) and 53% (f) of control

-        liver peroxisomal 13-oxidation was increased to 236% (m) and 123% (f) of control

-        liver microsomal content of cytochrome P450 isoenzyme CYP1A2 was decreased to 47% Cm) and 73% (f) of control

-        liver microsomal content of an unknown cytochrome P450 isoenzyme recognized by a monoclonal anti-CYP2B1/CYP2B2 antibody was decreased to 54% (m) and 41% (f) of control

-        liver microsomal content of cytochrome P450 isoenzyme CYP3A1 was increased to 124% (m) and 257% (f) of control and that of cytochrome P450 isoenzyme CYP3A2 was decreased to 46% (m) of control

-        liver microsomal contents of cytochrome P450 isoenzymes CYP4A1/3 and CYP4A2 were increased to 236% (m) and 207% (m) of control, respectively, and liver microsomal contents of cytochrome P450 isoenzymes CYP4A1/3 were decreased to 77% (f) of control.

Effects observed at 300 ppm (males: m; females: f):

-        liver porphyrin content was increased to 200% (m) and 200% (f) of control

-        plasma porphyrin content was increased to 198% (m) and 211% (f) of control

-        urinary porphyrin content (nmol/ml urine) was increased to 553% (m) and 375% (f) of control. Total urinary amount of porphyrin (nmol/24 h urine) was increased to 336% (f) of control

-        fecal porphyrin content (nmol/g wet feces) was increased 1184% (m) and 854% (f) of control. Total fecal amount of porphyrin (nmol/24 h feces) was increased to 990% (m) and 848% (f) of control

-        liver microsomal 7-methoxyresorufin 0-demethylase activity was decreased to 68% (f) of control

-        liver microsomal 7-ethoxyresorufin 0-deethylase activity was decreased to 68% (f) of control liver microsomal 7-pentoxyresorufin 0-depentylase activity was decreased to 69% (f) of control

-        liver microsomal content of cytochrome P450 isoenzyme CYP1A2 was decreased to 58% (m) and 79% (f) of control

-        liver microsomal content of an unknown cytochrome P450 isoenzyme recognized by a monoclonal anti-CYP2B1 /CYP2B2 antibody was decreased to 72% (m) and 63% (f) of control

- liver microsomal contents of cytochrome P450 isoenzymes CYP4A1/3 and CYP4A2 were increased to 168% (m) and 150% (m) of control.

Upon subchronic dietary administration to rats, the test substance interfered with heme biosynthesis with the consequence of dose-dependent, pronounced porphyria and a decreased activity of various isoenzymes of the hepatic microsomal cytochrome P450 system. A weak potency of the test article to act as a liver peroxisome proliferator was observed in high dosed males.