Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.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

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A reliable Ames test according to Japanese "Guidelines for Screening Mutagenicity Testing Of Chemicals" was negative in all strains with and without metabolic activation. This result was supported by a non-reliable Ames test (RL4 secondary source) and a non-reliable umu-test.



2-chlorotoluene was also tested in a relibale in vitro chromosome aberration study in mammalian cells using Chinese hamster ovary (CHO) cells. The test was negative with and without metabolic activation. The test has some limitations (e.g., only 100 metaphase cells were evaluated). Therefore, the in vivo test chromosome aberration in bone marrow cells from rats exposed to 2-chlorotoluene is used to support the results from the in vitro assay. This test was also negative.


The test substance was also tested in a relibale mouse lymphoma assay with and without metabolic activation. All experiments were negative (with and without metabolic activation), the test material, orthochlorotoluene, did not induce significant increases in the mutant frequency at the TK locus in L5178Y mouse lymphoma cells. The results are supported by a second, non-reliable (RL4, in vitro, secondary source)  mouse lymphoma forward mutation assay. The test material was assayed from 25 nl/ml to 90 nl/ml and a wide range of toxicities was induced without inducing significant dose-dependant increases above the background mutant frequency.


 


 


 


Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 18 February 1982 to 28 June 1982
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Colony sizing not reported
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
TK locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: adult, male Sprague Dawley ratsm, Aroclor 1254 induced
- method of preparation of S9 mix: prepared according to method of the laboratory
- concentration or volume of S9 mix and S9 in the final culture medium: 0.3 nl/10mL
Test concentrations with justification for top dose:
without metabilit activation:
Experiment 1: 1.95, 3.91, 7.81, 15.6, 31.3 nl/ml
Experiment 2: 7.5, 10, 15, 20, 30, 40 nl/ml

with metabolic activation:
Experiment 1: 3.91, 7.81, 15.6, 31.3, 62.5 nl/ml
Experiment 2: 10, 15, 20, 30, 40, 60 nl/ml
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: ethylmethane sulfonate; dimethylnitrosamine
Remarks:
ethylmethane sulfonate is used as positive control substance for assay without metabolic activation
dimethylnitrosamine is used as positive control substance for assay with metabolic activation
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of independent experiments: 4 (trial 3 was terminated due to contamination)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 - 3 days
- Selection time: 3 x 10E 6 cells for each selected dose are seeded in soft agar plates with selection medium and resistant (mutant) colonies are counted after 10 days incubation.
- Method used: agar
- selective agent: -bromo-2' deoxyuridine (BrdU), selectin medium contains 100 µg/ml BrdU

METHODS FOR MEASUREMENT OF CYTOTOXICITY
Relative cytotoxicities is expressed as the reduction in growth compared to the growth of untreated cells. This is used to select seven to ten doses in the range of 0 to 50-90% reduction in the 24-hour growth.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
To determine the actual number of cells capable of forming colonies, a portion of the cell suspension is also cloned in normal medium (nonselective). The ratio of resistant colonies to total viable cell number is the mutant frequency.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: without: 40 nl/ml; with: 60 nl/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Without metabolic activation , the test material was cytotoxic at 62.5 nl/ml. Five treatments from 1.95 nl/ml to 31.3 nl/ml were therefore chosen for mutant analysis in an attempt to obtain a wide range of toxic action. None of the assayed treatments induced a mutant frequency that exceeded the minimum criterion of 31.7x10 E-6. However, only moderate toxicities were induced (percent relative growths, 62.1% to 39.9%). Since it is preferable to include in the evaluation results from treatments that induced high toxicities (percent relative growths, 10% to 15%), another assay was performed.
In the second experiment without metabolic activation, the test material was assayed from 7.5 nl/ml to 40 nl/ml with small increments between concentrations. The assayed treatments induced percent relative growths ranging from 58.3% to 19.3% which included moderate toxicites. A sharp toxicity curve prevented analysis of highly toxic treatments; the next highest concentration (60 nl/ml) was lethal. In order for a treatment to be considered mutagenic in this assay a mutant frequency exceeding 38.5x10 E-6 was required and the assayed treatments induced mutant frequencies ranging from 10.9x10E- 6 to 23.7x10E- 6 . Since no evidence for mutagenic activity activity was observed, the test material was considered nonmutagenic without activation at oncentrations that approached lethality.
In the presence of metabolic activation, the test material was assayed from 10 nl/ml to 60 nl/ml. The minimum criterion for mutagenicity in this assay was a mutant frequency exceeding 52.4x 10E-6 . None of the assayed treatments induced this level of mutant action. The observed toxicities ranged from nondetectable to moderate (percent relative growths, 127.1% to 23.8%). High toxicities could not be analyzed because of a sharp toxicity curve (80 nl/ml was excessively toxic).
The test material was therefore considered nonmutagenic with activation at treatments that approached excessive toxicity. In the assays used in this evaluation, the average cloning efficiencies for the solvent and untreated negative controls varied from 80.1% and 84.4% without activation to 76.8% with activation which demonstrated good cloning conditions for the assays. The negative control mutant frequencies were all in the normal range and the positive control compounds yielded normal mutant frequencies that were greatly in excess of
the background.
For details on results see illustration below.
The test material, orthochlorotoluene, did not induce significant increases in the mutant frequency at the TK locus in L5178Y mouse lymphoma cells. The test material was assayed up to 40 µl/ml without activation and 60 nl/ml with activation, and nondetectable to moderate toxicities were induced. Highly toxic treatments were not available for analysis because of a sharp toxicity curve. None of the assayed treatments induced a significant increase in the background. The test material is therefore considered inactive with and without metabolic activation in the mouse lymphoma forward mutation assay at concentrations that approached excessive toxicity.
Conclusions:
In an mammalian cell gene mutation assay performed similar to OECD test guideline 476 with mouse lymphoma L5178Y cells under GLP conditions, the test substance was negative in two separate experiments with and without metabolic activation.
Executive summary:

2-chlorotoluene was tested in the mouse lymphoma assay with and without metabolic activation (Aroclor-induced rat liver S9 mix). Two separate experiments with concentrations of  0, 1.95, 3.91, 7.81, 15.6, 31.3 nl/ml (experiment 1 and 0, 7.5, 10, 15, 20, 30, 40 nl/ml (experiment 2, both without metabilit activation) and 0, 3.91, 7.81, 15.6, 31.3, 62.5 nl/ml (experiment 1) or  0, 10, 15, 20, 30, 40, 60 nl/ml (experiment 2, both with metabolic activation) were performed.


All experiments were negative (with and without metabolic activation), the test material, orthochlorotoluene, did not induce significant increases in the mutant frequency at the TK locus in L5178Y mouse lymphoma cells. The test material was assayed up to 40 µl/ml without activation and 60 nl/ml with activation, and nondetectable to moderate toxicities were induced. Highly toxic treatments were not available for analysis because of a sharp toxicity curve. None of the assayed treatments induced a significant increase in the background. The test material is therefore considered inactive with and without metabolic activation in the mouse lymphoma forward mutation assay at concentrations that approached excessive toxicity.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 28 April 1982 to 2 June 1982
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: in accordance with guideline
Principles of method if other than guideline:
2-chlorotoluene was tested in the chromosome aberration assay using Chinese hamster ovary cells. The assay was conducted both in the absence and presence of an aroclor-induced S-9 activation system at dose levels of 0.83, 2.50, 8.33, 25.00, 83.33 and 250 nl/ml for the non-activated study and for the S-9 activated study. Metaphase cells were collected at 2.0 to 2.5 hours after treatment for microscopic evaluation.
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
no data
Species / strain / cell type:
other: Chinese Hamster Ovary (CHO) cells
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: obtained from Dr. S. Wolff's laboratory, University of California, San Francisco, and cloned in Dr. A. Bloom's laboratory, Columbia University, New York.
- Normal cell cycle time (negative control): 12 - 14 h

MEDIA USED
- McCoy's 5a medium supplemented with 10% fetal calf serum, (FCS), glutamine, and antibiotics.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: purchased from Litton Biological products, Inc.
- method of preparation of S9 mix: Aroclor 1254 treated male Fisher rats
Test concentrations with justification for top dose:
With and without metabolic activation:
0, 0.83, 2.5, 8.33, 25.0, 83.33, 250.0 nl/ml
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: without S9: mityomycin c; with S9 mix: cyclophosphamide
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: single
- Number of independent experiments: one

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 1.5 x 10 E6 cells per 75 cm² flask in 10 ml medium (24 h before test substance was added)
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
Without metabolic activation: 8,5 - 10 h, with metabolic activation: 2 h

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): colcemid at a final concentration of 0.1 µg/ml for 2.5 h
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): metaphase cells wer collected by mitotic shake-off. Cells were swollen with 0.075 M KCl hypotonic solutio, washed 3 times in fixative solution (methanol : acetic acid, 3:1) dropped onto slides and
- Standard forms were used to score and record gaps, breaks, fragments and
reunion figures, as well as numerical aberrations such as polyploid cells.
The aberrations scored include:
chromatid gap
chromatid break
chromosome gap
chromosome break
chromatid deletion
chromatid fragment
acentric fragment
triradial
quadriradial
Statistics:
Statistical analysis emplayed the student t-test
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: 250 nl/ml: toxic level
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Without Metabolit Activation:
The test substance was severely toxic at 250 nl/ml, some reduction in cell growth was noted at 83.3 nl/ml. The frequencies of aberrations in the negative and solvent control cultures are within the normal background range for this laboratory, and the positive control compound, Mitomycin C, induced a significant amount of chromosome damage, including the chromatid interchanges (triradials and quadriradials) typical of chemical clastogenesis. There was no significant increase in chromosome damage in the cultures treated with o-chlorotoluene up to the rather toxic dose of 83.3 nl/ml. The very slight increases are not statistically significant, and there was no increase in the proportions of cells bearing aberrations. There was no evidence for a positive dose relation, and overall the result is negative.

With the Metabolic Activation :
There was severe toxicity at 250 nl/ml. There was reduced cell growth and division also at 83.3 nl/ml. The aberration frequencies in the negative and solvent controls are within normal background limits for this laboratory; while the number of aberrations in the negative control is at the upper limit of the historical range, the percentage of cells bearing aberrations (5%) is normal and four of the aberrations (double minute fragments) were contributed by only one cell. The large amount of damage in the positive control culture demonstrates the effective activation of the cyclophosphamide by the S9 system. There was no increase in aberrations in the test cultures exposed to o-chlorotoluene.

Results without metabolic activation:











































































TreatmentCells scoredNo. of
aberrations per cell
% cells
with abberations
% cells
with >1 abberations
Negative control (medium)100>0.066.02.0
Negative control (DMSO)1000.054.01.0
Positive control1000.17**16.01.0
0.83 nl/ml1000.096.02.0
2.5 nl/ml100>0.033.01.0
8.33 nl/ml100>0.086.02.0
25.00 nl/ml100>0.022.01.0
83.33 nl/ml100>0.066.03.0
250 nl/ml#0---

**Signiflcantly greater than solvent control. p<O. 01


# toxic level


Results with metabolic activation:











































































TreatmentCells scoredNo. of
aberrations per cell
% cells
with abberations
% cells
with >1 abberations
Negative control (medium)100>0.095.02.0
Negative control (DMSO)1000.033.00.0
Positive control1000.25**18.05.0
0.83 nl/ml100>0.043.03.0
2.5 nl/ml100>0.044.01.0
8.33 nl/ml100>0.033.02.0
25.00 nl/ml1000.011.00.0
83.33 nl/ml1000.022.00.0
250 nl/ml#0---

**Signifilcantly greater than solvent control. p<O. 01


# toxic level


 

Conclusions:
In an in vitro chromosome aberration study in mammalian cells performed in CHO cells under GLP conditions, the test substance was negative with and without metabolic activation.
Executive summary:

2-chlorotoluene was tested in an in vitro chromosome aberration study in mammalian cells using Chinese hamster ovary (CHO) cells. The assay was conducted both in the absence and presence of an aroclor-induced S9 activation system at dose levels of 0, 0.83, 2.50, 8.33, 25.00, 83.33 and 250 nl/ml for the non-activated study and for the S9 activated study. Metaphase cells were collected at 2.0 to 2.5 hours after treatment for microscopic evaluation.


Negative results were obtained with and without metabolic activation  up to the highest concentration that could be evaluated (severe cytotoxicity was observed at 250 nl/ml). The positive control substances yielded the expected results.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Principles of method if other than guideline:
Preincubation method according to Ames, Mutat. Res. 31, 347 (1975), Maron, Mutat. Res. 113, 173 (1983)
The test compound dissolved in 0.05 or 0.1 ml of solvent was supplemented with 0.5 ml of S9mix (metabolic activation method) or O.1 M phosphate buffer pH 7.4 (direct method) and 0.1 ml of tester strains which had been cultured in nutrient broth. The mixture was incubated for 20 min at 37°C, then rapidly mixed with 2 ml of molten top agar containing 0.05 µmol/ml of L-histidine and biotin for the Salmonella test. In the E. coli test 0.05/1 mol/ml of L-tryptophan was used instead of L-histidine and biotin. Then the top agar mixture was rapidly poured onto a 30 ml of Vogel-Bonner minimal agar plate. All plates were incubated for 48 hours at 37°C and the numbers of revertant colonies were scored.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: Salmonella typhimurium TA100, TA102, TA104, TA1535, TA98, TA1537, TA1538, Escherichia coli WP2uvrA, Escherichia coli WP2uvrA/pKM101
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Experiment
1) With and without metabolic activation (S. thyphimurium TA100, TA1535, TA98, TA1537, TA1538 and E.coli WP2 uvrA):
0, 5, 10, 20, 50, 100, 200, 500, 1000 µg/plate

2) With and without metabolic activation (S. typhimurium: TA100, TA1535, TA98, TA1537, TA102, TA104, E.coli WP2 uvrA and WP2uvrA/pKM101):
0, 0.0763, 0.305, 1.22, 4.88, 19.5, 78.1, 313, 1250, 5000 µg/plate

3) With and without metabolic activation (S. typhimurium: TA100, TA1535, TA98, TA1537, E.coli WP2 uvrA):
0, 2.44 (only -S9), 4.88 (only -S9), 9.77, 19.5, 39.1, 78.1, 156, 313 (only +S9), 625 (only + S9) µg/plate

4) With and without metabolic activation (S. typhimurium: TA102, TA104, E.coli WP2uvrA/pKM101):
0, 0.0763, 0.305, 1.22, 4.88, 19.5, 78.1, 313, 1250, 5000 µg/plate

5) With and without metabolic activation (S. typhimurium: TA102, TA104, E.coli WP2uvrA/pKM101):
0, 0.61(only -S9), 1.22 (only -S9), 2.44 (only -S9), 4.88 (only -S9), 9.77, 19.5, 39.1, 78.1 (only +S9), 156 (only +S9), 313 (only +S9), 625 (only + S9) µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: without activation: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, sodium azide, 9-aminoacridine, 2-nitrofluorene, 4-nitroquinoline-N-oxide, bleomycin, pyruvic aldehyde; with activation: 2-amino anthracene
Details on test system and experimental conditions:
Ames test
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 3
- Number of independent experiments 5

METHOD OF TREATMENT/ EXPOSURE:
- Preincumation method

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 min
- Exposure duration/duration of treatment: 48 h at 37 °C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- For determination of the toxicity of test compounds, the growth of the background lawn of tester strains an each plate was examined under a stereo microscope. This background growth is result of the addition of small amounts of histidine and biotin or tryptophan, and consists of micro colonies visible with aid of the stereo microscope. Bactericidal compounds lead to a decrease in the number of these micro colonies and to an increase in thier diameter.
When a toxicity was observed, an asterisk was placed to the right of the number of the colonies expressed in the data sheet.
Evaluation criteria:
Two-hold rule criteria was used for data evaluation (Ames et al., 1975). The chemicals are considered to be mutagenic when a dose-related increase in revertant colonycount is observed and the number of revertant colonies per plate with the test substance is more than twice that of the negative control (solvent control) and when a reproducibility of test result is observed.

Mutagenic potency was calculated by following equation and maximum value of
mutagenic potency was expressed as a specific acitivity on the data sheet: mutagenic potency (induced revertants / mg test substance) = (number of induced revertants on the dose X — number of revertant on the solvent control) / mg of test chemical on the dose X.
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxic at >=500 µg/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxic at >=1000 µg/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxic at >=500 µg/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cyctotoxic at >=500 µg/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxic at 1000 µg/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxic at >=500 µg/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxic at >=500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Results above only shown for experiement 1). The other experiments supported the results shown above: results for all strains and concentrations were negative. Since cytotoxicity was abserved for all strains starting at 500 or 1000 µg/plate concentrations higher than 1000 µg/plate could not be evaluated. For details on all experiments see attached illustration.

The positive controls were functional.


For details on results see attached PDF file with tables.


 

Conclusions:
In an Ames test according to Japanese "Guidelines for Screening Mutagenicity Testing Of Chemicals" the test substance was tested at concentrations up to 1000 µg/plate. Depending on the bacteria strain, cytotoxicity was observed between 500 and 1000 µg/plate with and without metabolic activation in all strains.
Under the conditions of the test, the test substance was negative in all strains.
Executive summary:

In an Ames test according to Japanese "Guidelines for Screening Mutagenicity Testing Of Chemicals" performed under GLP conditions  the test substance was tested at concentrations up to 1000 µg/plate in Salmonella thyphimurium strains TA100, TA1535, TA98, TA1537, TA1538 and E.coli WP2 uvrA. DMSO was selected as vehicle. Depending on the bacteria strain, cytotoxicity was observed between 500 and 1000 µg/plate with and without metabolic activation in all strains. The positive control substances resulted in the expected responses.
Under the conditions of the test, the test substance was negative in all strains.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

To support the negative results obtained in an in vitro chromosome aberration study in mammalian cells with Chinese hamster ovary (CHO) cells, an in vivo test for chromosome aberration in bone marrow cells from rats exposed to 2-chlorotoluene is used. This in vivo test was also negative.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Remarks:
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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
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:
yes
Type of assay:
mammalian bone marrow chromosome aberration test
Species:
rat
Strain:
Sprague-Dawley
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.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- 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:

ENVIRONMENTAL CONDITIONS
Sanitary cages and bedding were used.
Route of administration:
oral: gavage
Vehicle:
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
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:
yes
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.
Statistics:
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
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Positive control: valid

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.

Conclusions:
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information


In an Ames test according to Japanese "Guidelines for Screening Mutagenicity Testing Of Chemicals" performed under GLP conditions (RL 1 ) the test substance was tested at concentrations up to 1000 µg/plate in Salmonella thyphimurium strains TA100, TA1535, TA98, TA1537, TA1538 and E.coli WP2 uvrA. DMSO was selected as vehicle. Depending on the bacteria strain, cytotoxicity was observed between 500 and 1000 µg/plate with and without metabolic activation in all strains. The positive control substances resulted in the expected responses. Under the conditions of the test, the test substance was negative in all strains. This result was fully supported by a non-reliable Ames test also with and without metablic activation in several bacteria strains (RL4, secondary source) and a non-reliable umu-test.



2-chlorotoluene was tested in an in vitro chromosome aberration study (RL 2, in accordance with guideline) in mammalian cells using Chinese hamster ovary (CHO) cells. The assay was conducted both in the absence and presence of an aroclor-induced S9 activation system at dose levels of 0, 0.83, 2.50, 8.33, 25.00, 83.33 and 250 nl/ml for the non-activated study and for the S9 activated study. Metaphase cells were collected at 2.0 to 2.5 hours after treatment for microscopic evaluation.


Negative results were obtained with and without metabolic activation  up to the highest concentration that could be evaluated (severe cytotoxicity was observed at 250 nl/ml). The positive control substances yielded the expected results. The test has some limitations (e.g., only 100 metaphase cells were evaluated). Therefore, the in vivo test chromosome aberration in bone marrow cells from rats exposed to 2-chlorotoluene is used to support the results from the in vitro assay. in this assay (RL 2, study well documented, meets generally accepted scientific principles, acceptable for assessment) 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 bonw marrow was harvested at various times after treatment with colchicine. The test was negative.


2-chlorotoluene was tested in the mouse lymphoma assay (RL 2, similar to guideline) with and without metabolic activation (Aroclor-induced rat liver S9 mix). Two separate experiments with concentrations of  0, 1.95, 3.91, 7.81, 15.6, 31.3 nl/ml (experiment 1 and 0, 7.5, 10, 15, 20, 30, 40 nl/ml (experiment 2, both without metabolic activation) and 0, 3.91, 7.81, 15.6, 31.3, 62.5 nl/ml (experiment 1) or  0, 10, 15, 20, 30, 40, 60 nl/ml (experiment 2, both with metabolic activation) were performed.


All experiments were negative (with and without metabolic activation), the test material, orthochlorotoluene, did not induce significant increases in the mutant frequency at the TK locus in L5178Y mouse lymphoma cells. The test material was assayed up to 40 µl/ml without activation and 60 nl/ml with activation, and nondetectable to moderate toxicities were induced. Highly toxic treatments were not available for analysis because of a sharp toxicity curve. None of the assayed treatments induced a significant increase in the background. The test material is therefore considered inactive with and without metabolic activation in the mouse lymphoma forward mutation assay at concentrations that approached excessive toxicity. The results are supported by a second, non-reliable (RL4, in vitro, secondary source)  mouse lymphoma forward mutation assay. The test material was assayed from 25 nl/ml to 90 nl/ml and a wide range of toxicities was induced without inducing significant dose-dependant increases above the background mutant frequency.


 

Justification for classification or non-classification

From the available data a classification according to EU Regulation 1272/2008 (CLP) is not justified because all genetic toxicity tests were negative.