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Toxicological information

Specific investigations: other studies

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Administrative data

Endpoint:
hepatotoxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From July 1989 to November 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test protocol according to generally accepted scientific standards and described with adequate details

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
no guideline required
Principles of method if other than guideline:
The method used allowed to determine if the substance induced cytochrome P450 enzymes and if it was a hepatic peroxysome proliferator in the rat
GLP compliance:
not specified
Type of method:
in vivo
Endpoint addressed:
repeated dose toxicity: oral

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Test was performed with dibenzyltoluene, new name: dibenzylbenzene, ar-methyl derivative

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, France
- Age at study initiation: approximately 7 weeks old
- Weight at study initiation:
Mean initial bodyweight for males was 185 g
Mean initial bodyweight for females was 160 g
Number of animals: 6 males + 4 females
- Diet (e.g. ad libitum): a complete commercial diet ad libitum
- Water (e.g. ad libitum): tap-water through automatic waterers ad libitum
- Acclimation period: at least 1 week before beginning treatment


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 60 +/- 10
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 02 August To: 13 December 1989

Administration / exposure

Route of administration:
other: gavage
Vehicle:
other: 10% aqueous gum arabic solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The compound was prepared extemporaneously as a suspension in a 10% aqueous gum arabic solution

VEHICLE
- Justification for use and choice of vehicle (if other than water): no
- Concentration in vehicle: 10 %
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were carried out during the study to control the concentration of suspensions at approximately 3 months, 4 months and 4 months and 2 weeks after the beginning of the treatment period.
Duration of treatment / exposure:
Exposure period: at least 120 day(s)
Frequency of treatment:
daily
Post exposure period:
No
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 5, 50 and 500 mg/kg bw/day for peroxysome proliferation; 0 and 500 mg/kg bw/d for enzyme activity.
Basis:
nominal conc.
No. of animals per sex per dose:
10 (6 males + 4 females)
Control animals:
yes, concurrent vehicle
Details on study design:
- After having been removed and weighed, liver were sampled into 3 pieces of 1-3 g and frozen at -80°C until enzymatic analysis.
- Preparation of microsomal fraction for determination of enzyme activity:
Frozen liver samples are thawed at 4°C in buffer A (Tris acetate 0.1 M, KCl 0.1 M, EDTA 1 mM, 2,6-di-tert-butyl-4-methyl-phenol 2.3 10E-5 M).
After mixing with an Ultra-Turrax and homogenized with a Potter-Elvehjem tissue grinder, they are centrifuged at 9000 x g for 20 minutes.
The supernatant is collected and centrifuged at 105000 x g for 60 minutes. Protein pellet are then resuspended in 30 ml of buffer B (potassium
pyrophosphate 0.1M, EDTA 1 mM and 2,6-di-tert-butyl-4-methyl-phenol 2.3 10E-5 M). The suspension was centrifuged at 105000 x g for
90 minutes.
Protein pellet are resuspended in buffer C (KH2PO4 0.1M and EDTA 1 mM).
- Preparation of rat liver homogenates for peroxisome proliferation assessment:
Liver pieces were homogenized with a Potter-Elvehjem tissue grinder in a buffer consisting of Tris-KCl adjusted to pH 7.4. Liver homogenates were centrifuged at 4°C and 600 x g for 5 minutes. The supernatant was then centrifuged at 4°C and 15000 x g for 15 minutes. The pellet was resuspended in the Tris-KCl buffer to obtain a concentration of 20 to 30 mg/ml.

Examinations

Examinations:
- Liver weight was measured and liver/body weight was calculated.
- Determination of cytochrome P450 content:
Microsomal suspension was placed in spectrophotometer cuvette. Baseline was recorded then CO was carefully bubbled through the sample for
about one minute. The spectrum was then recorded between 350 and 500 nm and the cytochrome P450 concentration was calculated from the
optical density difference (450-490 nm) using the extinction molar coefficient of 91 mM-1.cm-1.
- Determination of cytochrome P450 activity by measuring:
. 7-ethoxyresorufin O-deethylation, as a marker of CYP1A activity
. d-benzphetamine N-demethylation, as a marker of CYP2B activity
. erythromycin N-demethylation, as a marker of CYP3A activity
. lauric acid 12-hydroxylation, as a marker of CYP4A activity
- Determination of peroxisome proliferation
. by palmitoyl-CoA-oxidase (PCO) measurement (results were then expressed as nanomoles oxidized palmitoyl-CoA per minute and per milligram
proteins)
. by peroxisome visualisation (method of Novikoff and Goldfisher with a JEOL 100S microscope) and counting (using a Commodore CBM 4032 analyser) in 2 males and 2 females. The cytoplasmic area studied for each animal was approximately 10.000 µm². Results were expressed as number of
peroxisomes per 100 µm².
Positive control:
No, but for discussion of the severity of the effects induced by the test substance, effect values obtained with reference substance in other studies
were presented for each enzyme activity evaluated:
- Cytochrome P450IA (7-ethoxyresorufin O-deethylation activity): 3-methylchloranthrene (x38 and x15 increased activity when compared to control animals, for males and females, respectively)
- Cytochrome P450IIB (d-benzphetamine N-demethylation activity): phenobarbital (x2.8 and x3.9 increased activity when compared to control
animals, for males and females, respectively)
- Cytochrome P450IIIA (erythromycin N-demethylation activity): dexamethasone (x6.6 and x14.7 increased activity when compared to control
animals, for males and females, respectively)
- Cytochrome P450IVA (lauric acid 12-hydroxylation activity: clofibratee (x3.3 increased activity for males when compared to control animals; females not determined).

Results and discussion

Details on results:
RESULTS:
Only CYP450-2B activity was increased and considered as toxicologically significant. Dibenzyltoluene was also found to induce a slight peroxysome proliferator.

Any other information on results incl. tables

RESULTS: Only CYP450-2B activity was increased and considered as toxicologically significant. LIVER WEIGHT: An increase in absolute liver weight was observed in males (+21%) and females (+11%). Relative weights (liver/body weight ratio) were also increased: +18% in males and +19% in females, but this increase was only statistically significant in males. EFFECTS ON CYTOCHROME P450 CONTENT: An increase in cytochrome P450 was observed in both males and females. However this increase was statistically significant in males only. EFFECTS ON DEMETHYLATION, DEETHYLATION AND HYDROXYLATION: - Effect on 7-ethoxyresorufin O-deethylation: dibenzyltoluene induced a statistically significant increase in 7-ethoxyresorufin O-deethylation in both male and female rats. 3-Methylcholanthrene, a potent inducer of CYP1A, triggered a 38- and 15-fold increase in males and females respectively. - Effect on d-benzphetamine N-demethylation: dibenzyltoluene induced a statistically significant increase in d-benzphetamine N-demethylation in both sexes. Phenobarbital, a specific and potent inducer of CYP2B, elicited a 2.8 to 3.9-fold increase of d-benzphetamine N-demethylation. - Effect on erythromycin N-demethylation: Neither male or female rats exhibited a significant induction of erythromycin N-demethylation. Following treatment of rats with dexamethasone, a potent inducer of CYP3A gene subfamily, a 6.6 to 14.7-fold increase in erythromycin N-demethylation was observed. - Effect on lauric acid 12-hydroxylation: dibenzyltoluene did not induce, in any sexes, a statistically significant dose-related increase of lauric acid 12-hydroxylation. Treatment of rats with clofibrate, a potent inducer of CYP4A gene subfamily, elicited a 3.5-fold increase. EFFECTS ON PEROXISOME PROLIFERATION: - Palmitoyl-CoA-oxidase activity: Jarylec DBT only induced a slight peroxisome proliferation in males (inducing factor: 1.8, compared to that of control) and in females (inducing factor: 1.5). Actually, mean PCO activity was 8.44 nmol/min/mg protein in exposed males (vs 4.58 in control males) and 9.88 nmol/min/mg protein in exposed females (vs 6.46 in control females). - Peroxisome count: Mean peroxisome count (number of peroxisome per 100 µm² cytoplasmic area) was slightly increased in treated group when compared to control group (8 vs 5.3).

Applicant's summary and conclusion

Conclusions:
Aalysis of hepatic enzymes activity in rats following a 4-month exposure to benzyltoluene at 500 mg/kg bw/d demonstrated that the test substance was a slight cytochrome P450 enzyme inducer. It was also found to induce the hepatic peroxysome proliferation. These results are consistent with increases in liver to body weight ration and hypertrophy of centrilobular hepatocytes that were also observed in rats following a 4-month exposure to dibenzyltolouene at 500 mg/kg bw/d.
Executive summary:

As part of the 4-month toxicity study (see section 7.5.1 Repeated dose toxicity oral) of dibenzyltoluene (JARYLEC DBT in the report) administered orally (0, 5, 50 and 500 mg/kg/day ) to Sprague-Dawley rats, the following hepatic enzyme activities were assessed at the end of the study in the high-dose group:

- Total cytochrome P450's,

- Cytochrome P450IA (7-ethoxyresorufin O-deethylation activity),

- Cytochrome P450IIB (d-benzphetamine N-demethylation activity).

- Cytochrome P450 IIIA (erythromycin N-demethylation activity),

- Cytochrome P450IVA (lauric acid 12 hydroxylation activity).

The hepatic peroxisome proliferation was determined in all groups by measurement of palmitoyl-CoA-oxidase activity and peroxisome counting using electron microscopy.

The exposure of rats during 4 months at 500 mg/kg bw/d showed:

- a slight but not toxicologically significant increase in P450IA-mediated reaction,

- an increase in P450IIB. Such increase, but a little bit higher is traditionally observed by phenobarbital induction. However, P450 reaction induced by phenobarbital is known to reverse rapidly. Modifications such as the increase in lver body weight ratio and the hypertrophy of the centrilobular hepatocytes are consistent with the enzymatic induction.

Assessment of peroxysome proliferation by measurement of the palmitoyl-CoA-oxidase activity was performed by oral route in rats treated with dibenzyltoluene at the dose-levels of 0, 5, 50 and 500 mg/kg bw/d during 4 months. This study showed that dibenzyltoluene induces only a slight peroxysome proliferation at 500 mg/kg bw/d (with no differences between sexes).