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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented publication which meets basic scientific principles

Data source

Reference
Reference Type:
publication
Title:
Demonstration of chlorobenzene-induced DNA demage in mouse lymphocytes using the single cell gel electrophoresis assay
Author:
Vaghef H & Hellman B
Year:
1995
Bibliographic source:
Toxicology 96, 19-28

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
The DNA demaging effect of chlorobenzene was investigated in peripheral lymphocytes and bone marrow cells from female mice using a gel electrophoresis assay for DNA from single cells ("the single cell gel electrophoresis assay") under alkaline conditions. The effect of chlorobenzene was studied both after single and repeated intraperitoneal injections of chlorobenzene
GLP compliance:
not specified
Type of assay:
other: DNA damage using single cell gel electrophoresis

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): chlorobenzene
- Analytical purity: no data

Test animals

Species:
mouse
Strain:
C57BL
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Alab, Solna, Sweden
- Weight at study initiation: 19-21 g
- Diet (e.g. ad libitum): standard pellet diet (EWOS AB, Södertälje, Sweden) ad libitum
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23°C
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light


Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: olive oil
Duration of treatment / exposure:
Single injection experiment: the duration of the exposure was 16 hours (from injection to sacrifice)
Repeated intraperitoneal injections experiment: the duration of the exposure was 3 days
Frequency of treatment:
Repeated intraperitoneal injections experiment: daily
Post exposure period:
No post exposure period
Doses / concentrations
Remarks:
Doses / Concentrations:
750 mg/kg body weight
Basis:
nominal conc.
No. of animals per sex per dose:
Single dose experiment: 6 animals
Repeated dose experiment ( 3 days): 3 animals
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: intraperitoneal injection.
- Doses / concentrations: 150 mg /kg bw
- Justification for choice of positive control(s): cyclophosphamide is well known genotoxic cytostatic agent.

Examinations

Tissues and cell types examined:
Peripheral blood lymphocytes and bone marrow. cells
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Chlorobenzene was diluted in olive oil immediately before being injected intraperitoneally (i.p.), either as a single injection (750 mg/kg bw), or repeatedly for 3 days (3 x 750 mg/kg bw). The genotoxic agent cyclophosphamide, freashly dissolved in physiolological saline , was used as a reference substance to verify the sensitivity of the test system. Control animals received olive oil intraperitoneally , either as a single injection (10µl/g bw), or repeatedly with 24 hours between each injection (3 x 10 µl/g). The animals were killed in a CO2-saturated atmosphere 16 hours after the (last) injection. Blood samples and bone marrow cells were collected from each mouse and put on ice immediately after sacrifice. The whole study was divided into three separate experiments with seven mice in each (2 controls, 2 animals receving the reference substance and 3 mice given chlorobenzene).

DETAILS OF SLIDE PREPARATION: The single cells (peripheral lymphocytes and bone marrow cells) were analyzed under alkaline conditions. Dakin fully frosted microscope slides were initially covered with melted 0.6% normal melting agarose. A mixture of cells and agarose (75µl/slide) was pipetted onto pre-coated microscope slide. After application of a new coverslip, the slides were allow ed to gel on ice as above. The coverslip was removed, and a third layer of melted 0.5% low melting-point agarose (85µl/slide; 37°C) without cells, was gently applied as above. After removal of the coverslip, the cells were lysed and the microscope slides were placed in an electrophoresis unit. After the electrophoreis , the slides were carefully neutralized (2 times) with 0.4 M Trizma base for 5 minutes. After application of a coverslip, each slide was observed at 250 x magnification.


METHOD OF ANALYSIS:
A semiautomatic image system was used for quantitative evaluation of the DNA demage. A typical image, as observed in the fluorescence microscope, usually contains several objetcs looking like "comets". Each comet is built up by heavily stained cell nucleus ("comet head") with or without a less fluorescent " tail" of migrating genetic material, probably representing DNA-fragments. Cells with increased DNA demage display an increased migration of DNA-fragments from the nucleus towards the anode. The extent of demage in an individual cell can then be quantified by measuring various parameters of the displacement between the tail and the comet head.
Three different head parameters were calculated for each thresholded image: head size (the diameter of the cell nucleus in pixels); head area (the total area of the cell nucleus in pixels), and the head average intensity (the average absolute intensity of cell nucleus). For each tail, data was gathered on 1) total area; 2) average absolute intensity; and 3) distance to the center position of the head. The program gives: a) the tail leght; b) the tail average intensity of all tail lenght; c) the total tail area; d) the percentage of DNA in the comet (TDNA); e) the tail distance (TDx; the distance in x-direction between the center position of the head and the center gravity of the tail) ; and f) the tail moment (i.e., the torsional moment which is derived by multplying TDNA with TDx). All calculations were based on absolute intensities, and units for length are given as "number of pixels".


Evaluation criteria:
To judge whether a specific treatment induced DNA demage a two-by-two table was constructed by classifyng the cells from the controls and the treated animals as either "negative " or "positive". The 75 percentile for the tail moment in the vehicle-treated control group was used as a cut-off point were classifed as "positive", and those with lower tail moments as "negative". A similar analysis was made using the tail moment corresponding to the 95 percentile among the controls as an alternative cut-off point.
Statistics:
Statistical significance was judged using one-tailed Fisher´s exact test for two-by-two tables, testing the hypothesis that the number of cells with demaged DNA was increased in mice given cyclephosphamide or chlorobenzene. The Kruskall-Wallis one-way analysis of variance by ranks was used totest whether the distribution of various tail characteristics differed between individual groups with identical treatments in separate expetiments. The one-tailed Kolmogorov-Smirnow two samples test was used to test the hypothesis that the various tail parameters were increased in cell populations isolated from the cyclophosphamide and chlorobenzene-treated animals. These analyses were made for the entire cell populations. For comparative purposes, the various tail parameters were also analysed using the one-tailed t-test or indipedent samples.

Results and discussion

Test results
Sex:
female
Genotoxicity:
other: There was evidence of chlorobenzene-induced DNA damage after 3 days of repeated exposure in peripheral lymphocytes, but no indications of such an effect in bone marrow cells. Thus. high-dose exposure to chlorobenzene is associated with DNA damage
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid

Any other information on results incl. tables

Table1 Percentage of peripheral lmphocytes and bone marrow cells with demaged DNA after administration of olive oil, ciclophosphamide or chlorobenzene.

 Treatment  Cut-off point (percentile for tail moment among control)     Peripheral lymphocytes     Bone marrow cells      
     Percentage of "positive cells"  Increase in comparison to    controls (percentage)   Percentage of "positive cells"     Increase in comparison to   controls (percentage)  
       mean ± S.E  [95% CI]     mean ± S.E   [95% CI]
 Controls  95  5  -  -  5  -  -
 olive oil  75  25  -  -  25  -  -
 Cyclophophamide  95  67 ***  62  ± 4  [54 -70]  14 **   9 ± 2  [3 -14]
 150 mg/kg  75  93 ***  68 ± 5  [59 -77]  40 ***  15 ±5  [6 -24]
Chlorobenzene   95  5  -  -  3  -  -
 1 x 750 mg/kg  75  25  -  -  16  -  -
 3 x 750 mg/kg  95  14 ***  8 ±2  [4 -13]  1  -  -
   75  49 ***  23 ± 4  [15 -32]  9  -  -

The results are presented as percentages of cells with tail moments exceeding the 75 and 95 percentiles for the tail moments among controls. The number of analyzed cells in each group varied between 180 and 360. For all groups of treatments, the viability of both the lymphocytes and bone marrow cells was over 95%. If a treatment was associated with an increase in the number of "positve" cells, the percentage of the mean increase +- S.E.M is given together with the 95 % confidence interval for the observed difference. Statistical signifcance was judged using the one-tailed Fisher´s exact test for two-by-two tables.

** P< 0.01; *** P < 0.001

Table 2 Tail parameters of peripheral lymphocytes and bone marrow cells from mice given olive oil, cyclophosphamide or chlorobenzene

 Treatment  Cell Type  Tail lenght  Tail length/Head size  Percentage DNA in tail  Tail moment
     Mean +-SE (median)   Mean +-SE (median)  Mean +-SE (median)   Mean +-SE (median) 
 Controls  Lymphocytes  53 ± 3 (40)  0.8 ± 0.03 (0.6)  0.7 ± 0.05 (0.3)  24 ± 2 (8)
 olive oil  Bone marrow cells  76 ± 4 (58)  1.1 ± 0.05 (0.8)  2.4 ± 0.20 (1.4)  105 ± 10 (44)
 Cyclophosphamide  Lymphocytes  120 ± 4 ** (114)  1.7 ± 0.06 ** (1.6)  4.7 ± 0.24 ** (4.1)  200 ± 12 ** (166)
 150 mg/kg   Bone marrow cells  105 ± 3 ** (102)  1.4 ± 0.04 ** (1.4)  4.5 ± 0.27 ** (2.9)  201 ± 15 ** (112)
 Chlorobenzene   Lymphocytes  50 ±2 (37)  0.7 ± 0.04 (0.5)  0.6 ± 0.05 (0.2)  21 ± 2 (4)
 1 x 750 mg /kg   Bone marrow cells  70 ± 4 (48)  1.0 ± 0.05 (0.8)  1.9 ± 0.23 (0.6)  79 ± 11 (23)
 3 x 750 mg/kg    Lymphocytes  58 ± 3 * (48)  0.8 ± 0.05 * (0.7)  1.3 ± 0.13 ** (0.7)  50 ± 5 ** (22)
    Bone marrow cells  60 ± 3 (47)  0.9 ± 0.04 (0.7)  1.2 ± 0.12 (0.7)  49 ± 6 (20)

The results are presented as mean values ± SEM, together with the corresponding median values for 6 control mice; 6 mice given cyclphosphamide; 6 mice given a sdingle dose of chlorobenzene, and 3 mice given repeated injection of chlorobenzene. The number of analyzed cells varied between 180 and 360 in each group. For all treatments, the viability of both the lymphocytes and the bone marrow cells was over 95%. Statistical significances were judged using one-tailed Kolmogorov-smirnov two-sample test.

* P < 0.05; ** P < 0.001

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): positive
Executive summary:

Vaghef & al., 1995.

The DNA damaging effect of chlorobenzene was investigated in peripheral lymphocytes and bone marrow cells from C57BL/6 female mice using a gel elctrophoresis assay for DNA from single cells ("the single cell gel electrophoresis assay") under alkaline conditions. The effect of chlorobenzene was studied both after single and repeated intraperitoneal injections of 750 mg/kg body weight in olive oil. The cytostatic agent cyclephosphamide (150 mg/kg, i.p) was used a reference substance, and vehicle-treated mice as controls. DNA demage was recorded 16 h after the (last) injection, using an automated computerized image analyses system specifically designed for the sinlge cell gel electrophoresis assay. There was evidence of chlorobenzene-induced DNA demage after 3 days of repeated exposure in peripheral lymphocytes, but no indications of such an affect in bone marrow cells. No effects were observed after single injection.

It is concluded that high-dose exposure to chlorobenzene is associated with genotoxicity to peripheral lymphocytes.