Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
other information
Study period:
2002-2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
α,2-dichlorotoluene
EC Number:
210-258-8
EC Name:
α,2-dichlorotoluene
Cas Number:
611-19-8
Molecular formula:
C7H6Cl2
IUPAC Name:
1-chloro-2-(chloromethyl)benzene
Details on test material:
Source: Clariant GmbH-Batch No. DEBG 047131-Purity: 99.6%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harla Winkelmann GmbH, Borchen, Germany
- Age at study initiation: male/female animals appr. 6 weeks
- Weight at study initiation: male animals: 167 - 198 g (mean: 180.8 g); female animals: 137 - 159 g (mean: 145.4 g)
- Assigned to test groups randomly: yes
- Housing: 5 animals per macrolon cage type IV on soft wood granulate
- Diet: rat/mice diet ssniff R/M-H, ad libitum (ssniff GmbH, Soest, Germany)
- Water: tap water in platic bottles, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 50 +/- 20
- Air changes (per hr): air-conditioned room
- Photoperiod (hrs dark / hrs light): 12 /12

IN-LIFE DATES: From: 04-Feb 2002 To: 06-Feb 2002

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle/solvent used: sesame oil
- Justification for choice of solvent/vehicle: stability in the sovent
- Concentration of test material in vehicle: 5, 15, 50 mg/mL
- Amount of vehicle: 10mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
One day before administration the test substance was dissolved in sesame oil at appropriate concentration. With regard to homogeneity, preparation was stirred until dosing had been completed. The positive control was dissolved in distilled water; final concentration: 4 mg/mL.
Duration of treatment / exposure:
Twice at an interval of 24 hours (except positive control with only one dose)
Frequency of treatment:
see "Duration of treatment / exposure"
Post exposure period:
24 h
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 150, 500 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Endoxan R (cyclophosphamide)
- Route of administration: oral
- Doses / concentrations: 40 mg/kg bw

Examinations

Tissues and cell types examined:
Polychromatic erythrocytes gained from femoral bone marrow.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Preliminary dose-range finding study; test item was applied to 3 males and 3 females at a dose of (i) 600, (ii) 400, and (iii) 500 mg/kg bw. Oral administration of 600 mg/kg bw resulted in mortality in one male and two female rats. No macroscopic findings were observed and as clinical signs decreased motor activity and cowering posture were noticed at all doses. Highest sublethal dose of 500 mg/kg bw was selected for the main study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):

DETAILS OF SLIDE PREPARATION:
Femoral bone marrow was extracted, suspended in fetal bovine serum and centrifuged. Supernatant was discarded and drops of mixed sediment were smeared onto slides. Slides were stained with May-Grünwald's and Giemsa solution and finally coated.

METHOD OF ANALYSIS:
2000 polychromatic erythrocytes were counted per animal (number of cells with micronuclei was recorded). Additionally, the ratio of polychromatic erythrocytes to 200 normochromatic erythrocytes was determined. Main parameter for the statistical analysis was the proportion of polychromatic erythrocytes with micronuclei out of the 2000 counted erythrocytes.
Evaluation criteria:
Proportion of polychromatic erythrocytes with micronuclei in the positive control is significantly higher than in the negative control (p<0.05).
A test substance producing no significant dose-related increase in the number of micronucleated polychromatic erythrocytes is considered as non-clastogenic.
Statistics:
An one-sided Wilcoxon test was used. A significance level of 5 % was adopted for all tests.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
after twice oral administration of 500 mg/kg bw
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
see below

Any other information on results incl. tables

Oral administration of 500 mg/kg bw resulted in the death of

one male out of 10 animals treated. This animal was replaced

and survived after treatment. The following signs of toxicity

were observed in the main study from 2 hours to 6 hours after

the second application: diarrhea, stilted gait and cowering posture.

The dissection of the animals revealed no test substance
related macroscopic findings.

Animals from the other dose groups (50 mg/kg bw, 150 mg/kg
bw) showed neither clinical signs of toxicity nor
macroscopic findings after dissection.

The bone marrow smears were examined for the occurrence of
micronuclei in red blood cells. The results are summarized
in Table 1.

The incidence of micronucleated polychromatic erythrocytes
in the dose groups with o-chlorobenzyl chloride (OCBC) was
within the normal range of the negative control groups (mean

of  micronucleated polychromatic erythrocytes per 2000 cells:

1.7 -4.9). No  statistically significant increase in
micronucleated polychromatic erythrocytes was observed.

The ratio of polychromatic erythrocytes to total erythrocytes

in both male and female animals differed less than 20 % from

the control value in all dose groups, but decreased dose

dependently indicating slight toxicity in the highest dose group.

From the results, it was concluded that OCBC did not cause

a substantial increase in micronucleated polychromatic
erythrocytes and is not clastogenic in the micronucleus test

in vivo under the conditions described in this study.

Table 1.Results 
========================================================
Sex     Dose         Poly/    Poly/   Poly      Poly 
        (mg/kg bw)   animal   Ery     with MN   with MN        
                     counted  Mean    Mean      Mean [%]
--------------------------------------------------------
male    0-control    2000     0.47    3.0       0.15
male    50           2000     0.54    4.0       0.20
male    150          2000     0.51    3.2       0.16
male    500          2000     0.43    2.8       0.14
male    40-Endoxan   2000     0.46    30.8*     1.54
--------------------------------------------------------
female  0-control    2000     0.49    3.0       0.15
female  50           2000     0.50    3.0       0.15
female  150          2000     0.50    3.2       0.16
female  500          2000     0.41    3.2       0.16
female  40-Endoxan   2000     0.40    22.8*     1.14
=======================================================
*= significantly different from control (p<0.05)

Applicant's summary and conclusion

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

In a bone marrow micronucleus assay Sprague-Dawley rats were treated orally with o-Chlorobenzylchloride (99.6 % a.i.); the vehicle was sesame oil. The micronucleus assay in male and female rats was conducted according to OECD TG 474 in compliance with GLP. A preliminary experiment showed that no death occurred at doses of 400 and 500 mg/kg bw while death (one out of three males and two out of three females) was observed at 600 mg/kg bw. Thus o-Chlorobenzylchloride was orally administered twice at an interval of 24 hours to the animals at 0, 50, 150 and 500 mg/kg bw. There were signs of toxicity in the dose group of 500 mg/kg bw: one out of ten animals died and the following clinical signs were observed 2 to 6 hours after the second treatment: diarrhea, stilted gait and cowering posture. All animals were sacrificed 24 hours after the second treatment, bone marrow cells harvested and exmined for micronuclei. The positive control induced the appropriate response. No statistically significant increase in the micronucleated polychromatic erythrocyte frequencies was observed in any dose groups, indicating that o-Chlorobenzylchloride is not clastogenic in vivo.