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Toxicological information

Specific investigations: other studies

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Administrative data

Endpoint:
mechanistic studies
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: - Accepable, well- documented publication/study report which meets basic scientific principles

Data source

Reference
Reference Type:
publication
Title:
A temporal study on the histopathological, biochemical and molecular responses of CCl(4)-induced hepatotoxicity in Cyp2e1-null mice.
Author:
Avasarala S, Yang L, Sun Y, Leung AW, Chan WY, Cheung WT, Lee SS.
Year:
2006
Bibliographic source:
Toxicology, vol. 228, no. 2-3, p. 310-22

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
- wild type SV/129/ter mice and Cyp2E1-null mice of the same strain were compared in their reaction to a single ip injection of 1.59 g/kg CTC
- adverse reactions were tested in time dependent manner by liver histopathology and analysis of serum aminotransferase activity and changes in gene expression
GLP compliance:
no
Type of method:
in vivo
Endpoint addressed:
basic toxicokinetics

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): carbon tetrachloride
- Physical state: liquid
- Analytical purity: 99.8 %
- provider: Riedel-de-Haen, Germany

Test animals

Species:
rat
Strain:
other: SV/129/ter
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: National Cancer Institute (National Institutes of Health, Bethesda, MD, USA)
- Age at study initiation: 3 months
- Weight at study initiation: 20 - 30 g
- Fasting period before study: not reported
- Housing: not reported
- Diet (e.g. ad libitum): mouse diet No. 5015 (PMI Nutrition lnc., St. Louis. MO, USA)
- Water (e.g. ad libitum): chlorinated tap water
- Acclimation period: 1 wk


ENVIRONMENTAL CONDITIONS
- Temperature (°C): not reported
- Humidity (%): not reported
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): not reported


IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- 10 % CTC in corn oil

ADMINISTRATION
- single injection of 1.0 ml/kg bw CTC (= 1.59 g/kg bw, = 10 ml/kg bw of total solution) intraperitoneal


VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
single treatment, observation period 48 h post treatment
Frequency of treatment:
single treatment
Post exposure period:
observation period 48 h post treatment
Doses / concentrations
Remarks:
Doses / Concentrations:
1.0 ml/kg bw (=1.59 g/kg bw)
Basis:
other: nominal injected ip
No. of animals per sex per dose:
5 - 12 males of wild-type and Cyp2E1-null mice respectively
Control animals:
yes, concurrent vehicle
Details on study design:
- after single dosage animals of the four groups (WT and Cyp2E1-null mice, treated and control animals) were killed after 2, 6, 12, 24 and 48 h

Examinations

Examinations:
- decaptivated of animals at certain endpoints, blood collected, livers excised, weighted and flashfrozen in liquid nitrogen and stored for RNA extraction
- determination of serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST)
- liver histological analysis with sections from the median liver lobe
- RNA extraction of frozen liver samples for mRNA differential display analysis and nothern blot analysis
Positive control:
yes, wild type mice on the level of animals, vehicle control on the level of treatment

Results and discussion

Details on results:
morphological level:
- no deaths occured in any treatment group
- no obvious effect on liver weights in the any treatment group within the observation period
- distinct color change (red to orange) in the CTC treated wild-type mice 24 and 48 h after treatment indicative of a fatty infiltration into the liver
histopathologic findings:
- no histologic abnormalities in the vehicle treated groups and the CTC treated Cyp2E1-null mice at any time point
- no histologic abnormalities in the CTC treated wild type mice sacrificed at timepoints 2, 6 and 12 h post treatment
- drastic histological damage in the CTC treated wild type mice sacrificed at timepoints 24 and 48 h post treatment: balloned cells in the
centrilobular region, collapsing hepatic sinusoids
biochemical parameters:
- no significant increase of serum aminotransferase levels (AST, ALT) at any time point in CTC treated Cyp2E1-null mice as compared to their
vehicle treated controls
- no significant increase of serum aminotransferase levels (AST, ALT) at timepoints 2 and 6 h post treatment in CTC treated wild-type mice as
compared to their vehicle treated controls
- drastic increase of serum aminotransferase levels (AST, ALT) at timepoints 12 to 48 h post treatment in CTC treated wild-type mice as
compared to their vehicle treated controls, peaking at 24 h post treatment (52.4 fold increase for ALT, 268 fold increase for AST)
gene expression analysis:
- significant changes in gene regulation between CTC treated Cyp2E1-null mice and wild-type mice 24 h post treatment
function of the regulated genes: liver cirrhosis (gene: Pah), apoptosis (gene: Bnip3), oxidalive stress (gene: Car3),
xenobiotic detoxification (gene: Cyp1a2), lipid metabolism (gene: ADKP), chemosensory signaling or tumorigenesis (gene: MUP II),
structural organization (genes: Tubb 2 and Actg), regeneration (gene: Chka) and inflamatory response (gene: IL-1RAcP)


Applicant's summary and conclusion

Executive summary:

The study Avasarala (2006) describes the cytochrome P450 enzyme Cyp2E1 to be the key enzyme in transformation of CTC into toxic metabolites.

Wild type SV/129/ter mice and Cyp2E1-null mice of the same strain were treated with CTC by a single intraperitoneal injection of 1 ml/kg/bw (=1.59 g/kg bw) in corn oil as vehicle. The time course of development of liver injury was followed by analysis of liver weights and color, histologic effects in the liver, biochemical examinations (serum levels of ALT and AST) and changes in gene expression at time points 2, 6, 12 24 and 48 h post treatment. All parameters indicative of liver toxicity were changed according to expectations in the wild type mice after CTC treatment while on the contrary all of these parameters remained in normal physiological ranges in the Cyp2E1-null mice treated with CTC. This highlights the importance of the action of Cyp2E1 on CTC for the development of the toxic potential of CTC in the mamalian organism.