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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 12 MARCH 1982 to 22 JUNE 1982
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference Type:
study report

Materials and methods

Principles of method if other than guideline:
Method: according to EMS, Toxicol. Appl. Pharmacol. 22, 269-275 (1972): 4 rats/sex/dose/treatment time, killed 6, 24 and 48 hrs after dosing.
Chromosome aberration frequencies were determined in the bone marrow cells from rats exposed to 2-chlorotoluene administered per os either as acute (single dose) or a subacute (five daily doses) exposure. Three doses (300 mg/kg bw, 100 mg/kg bw and 30 mg/kg bw) were investigated and marrow was harvested at various times after treatment with colchicine (according to EMS, Toxicol. Appl. Pharmacol. 22, 269-275 (1972))
GLP compliance:
Type of assay:
mammalian bone marrow chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
Details on test material:
purity not specified, or relating to pure substance in case of QSAR

Test animals

Details on species / strain selection:
This healthy, random bred strain has been selected to maximize genetic heterogeneity and at the same time assure access to a common source.
Details on test animals or test system and environmental conditions:
- Source: Charles River Breeding Laboratories Inc.
- Weight at study initiation: animals were weighted, weight not reported
- Assigned to test groups randomly: Animals were assigned to study groups at random according to LBI Standard Operating Procedures (SOP)
- Housing: group-housed up to 4 rats/cage
- Diet: commercial diet (Purina Laboratory Chow) ad libitum
- Water: ad libitum
- Acclimation period:

Sanitary cages and bedding were used.

Administration / exposure

Route of administration:
oral: gavage
corn oil
Details on exposure:
Prior to study initiation animals were weighed to calculate dose levels according to SOP Animal Weight Determination." The volume of test article administered was established using this method unless there was significant variation among individuals, in which case individual calculations were made. Animals were uniquely identified by ear punch. Dose or treatment groups were ident ified by cage card.
Duration of treatment / exposure:
experiment 1 (acute): single treatment
experiment 2 (subacute): five consecutive doses, 24 h apart
Frequency of treatment:
experiment 1: single dose
experiment 2: daily treatment
Post exposure period:
experiment 1: 6, 24 or 48 h
experiemnt 2: 24 h
Doses / concentrationsopen allclose all
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
0 mg/kg bw/day (nominal)
No. of animals per sex per dose:
experiment 1 (acute): 8 animals per dose group (4 males and 4 females) and post-exposure treatment duration
experiment 2: (subacute): 8 animals per dose group (4 males and 4 females)
Control animals:
Positive control(s):
Triethylenemelamine (TEM)
- Route of administration: single i.p. injection
- Doses / concentrations: - 1.0 and 0,75 mg/kbg bw


Tissues and cell types examined:
The adhering soft tissue and epiphyses of both tibiae were removed according to the method of Legator et al. (1969). The marrow was flushed from the bone and transferred to Hanks' balanced salt solution.
Routinely, 50 cell spreads were read for each animal. The location of cells bearing aberrations was identified by the use of coordinates on the mechanical stage. Amitotic index based on at least 500 cells counted was also recorded. It was calculated by scoring the number of cells in mitosis per 500 cells on each slide read.
Details of tissue and slide preparation:
Three to four hours prior to kill, the animals were injected IP with 4.0 mg/kg bw of colchicine. The animals were killed with CO2 at the times indicated.The marrow of the tibiae was collected by centrifugation and resuspended in 0.075M KCl.The centrifugation was repeated and the pellet was resuspended in fixative methanol:acetic acid, 3:1). The fixative was changed after 1/2 hour and the cells left overnight at 4 °C.
Cells in fixative were dropped onto glass slides and air-dried. Spreads were stained with 5% Giemsa at pH 6.8.
Slides were coded and scored for chromosomal aberrations. Standard forms were used to score and record gaps, breaks, fragments and reunion figures.
Evaluation criteria:
Gaps were not counted as significant aberrations. Open breaks were considered as indicators of genetic damage, as were configurations resulting from the repair of breaks. The latter included translocations, multiradials, rings, multicentrics, etc. Reunion figures such as these were weighted slightly higher than breaks
since they usually resulted from more than one break.

The number of aberrations per cell was also considered significant, cells with more than one aberration were considered to indicate more genetic damage than those containing evidence of single events. Consistent variations from the euploid number were also considered in the evaluation of mutagenic potential.

Frequently, one is unable to locate suitable metaphase spreads for each animal even after preparing additional slides. Possible causes for this to appear related to cytotoxic effects which alter the duration of the cell cycle, kill the cell, or cause clumping of the chromosomes. Additional information was gained from the mitotic index which also appeared to reflect cytotoxic effects.

In any event thp. type of aberration, its frequency and its correlation to dose in a given time period were considered in evaluating a test article as being mutagenically positive or negative.
Statistical analysis employed a Student´s t-test.

Statistical tests were run on
1. the number of structural aberrations per animal
2. the number of numerical aberrations per animal
3. the percentage of cells with structural aberrations per animal
4. the percentage of cells with two or more structural aberrations per animal
Statistical evaluation was done relative to concurrent negative controls

Results and discussion

Test results
Key result
no effects
Vehicle controls validity:
Negative controls validity:
not examined
Positive controls validity:
Additional information on results:
Positive control: valid

Any other information on results incl. tables

The test article did not induce significant increases in structural aberration frequency at any of the dose-level/kill-time combinations for either sex, when the dosed animals were compared to the corresponding negative controls.

For details see illustration below.

Applicant's summary and conclusion

Chromosome aberration frequencies were determined in the bone marrow cells from rats exposed to 2-chlorotoluene. The test was negative.
Executive summary:

Chromosome aberration frequencies were determined in the bone marrow cells from rats exposed to 2-chlorotoluene administered per os either as acute (single dose, experiment 1) or a subacute (five daily doses, experiment 2) exposure. Three doses (300 mg/kg, 100 mg/kg and 30 mg/kg) were investigated and marrow was harvested at various times after treatment (6, 24 or 48 h after treatment for experiment 1 and 24 h for experiment 2)  with colchicine. Negative (vehicle) and positive controls were run in parallel.

The test article did not induce significant increases in structural aberration frequencyat any of the dose-level/kill-time combinations for either sex.