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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The mutagenic activity of the test item was investigated in a reliable bacterial reverse mutation assay (Ames test; test strains used: S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2uvrA, according to OECD guideline 471)), in a reliable in vitro gene mutation study in mammalian cells (mouse lymphoma test in L5178Y cells according to OECD guideline 476) and in a reliable in vitro chromosome aberration test in CHO cells (according to OECD guideline 473). Negative results were obtained in these tests with and without metabolic activation, except an equivocal result in the gene mutation test in mammalian cells in vitro without metabolic activation.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 11 Jul 2003 to 7 Aug 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
February 1998
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 liver extract from Aroclor 1254-induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
Experiment 1:
0, 15, 50, 150, 500, 1500 and 5000 µg/plate for WP2 uvrA in the presence of metabolic activation
0, 5.0, 15, 50, 150, 500 and 1500 µg/plate for other test samples
Highest test concentrations were selected on the basis of a preliniary toxicity assay

Experiment 2:
0, 7.5, 25, 75, 200, 600, 1800 and 5000 µg/plate with tester strains TA98 and WP2 uvrA in the presence of S9 activation
0, 2.5, 7.5, 25, 75, 200 and 600 µg/plate for other test samples
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: based on solubility of the test article and compatibility with the target cells
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene for TA98, Sodium azide for TA100 and TA1535, 9-aminoacridine for TA1537, Methyl methanesulfonate for WP2 uvrA
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
all strains with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): approx. 10exp9 cells/ml

DURATION
- Exposure duration: 48-72 h
- Fixation time (start of exposure up to fixation or harvest of cells): 48-72 h

DETERMINATION OF CYTOTOXICITY
- Method: >50 % reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count;
or moderate reduction in the background lawn
Rationale for test conditions:
Highest test concentrations selected on the basis of a preliniary toxicity assay
Evaluation criteria:
For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article. Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2.0-times the mean vehicle control value.
Statistics:
not specified
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Cytotoxicity (reduction in the mean number of revertants per plate or moderate reduction in the background lawn) was observed beginning at 150, 500, 1500 or 5000 µg/plate in experiment 1 and at 75, 200, 600, 1800 or 5000 µg/plate in experiment 2. No precipitate was observed in both experiments.
Conclusions:
Under the conditions of this study, the test item was not mutagenic to bacteria (Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA)
Executive summary:

The test article, 1-Chloro-2,3-dimethylbenzene, was tested in the Bacterial Reverse Mutation Assay with an Independent Repeat Assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9, using the plate incorporation method. In the initial mutagenicity assay, no positive mutagenic response was observed. The dose levels tested were 0, 15, 50, 150, 500, 1500 and 5000 µg/plate with tester strain WP2 uvrA in the presence of S9 activation and 0, 5.0, 15, 50, 150, 500 and 1500 µg/plate with all other test conditions. Toxicity was observed beginning at 150, 500, 1500 or 5000 in per plate. No precipitate was observed. In the independent repeat mutagenicity assay, no positive mutagenic response was observed. The dose levels tested were 0, 7.5, 25, 75, 200, 600, 1800 and 5000 µg/plate with tester strains TA98 and WP2 uvrA in the presence of S9 activation and 2.5, 7.5, 25, 75, 200 and 600 µg/plate with all other test conditions. Toxicity was observed beginning at 75, 200, 600, 1800 or 5000 µg/plate. No precipitate was observed. Under the conditions of this study, test article 3-Chloro-o-xylene was concluded to be negative in the Bacterial Reverse Mutation Assay with an Independent Repeat Assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 10 Jul 2003 to 17 Jul 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
February 1998
GLP compliance:
yes
Type of assay:
other: gene mutations in mammalian cells in vitro
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 liver extract from Aroclor 1254-induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
0, 5, 10, 20, 40, 60, 80, 100 µg/ml in all experiments
Highest concentration based on pretest, where concentrations of >/= 140 µg/ml produced > 50 % growth reduction
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: based on solubility of the test article and compatibility with the target cells
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION

- Exposure duration: 4 and 20 h without metabolic activation, 4 h with metabolic activation
- Expression time (cells in growth medium): 0 (at 20 h exposure), 16 h (4 h exposure)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 h for all experiments

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): if possible, minimum of 200 (100 per duplicate plates)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
The percentage of cells with chromosome aberrations in the positive control must be statistically increased (p<0.05, Fisher's exact test) relative to the solvent control.
Statistics:
Statistical analysis of the percent aberrant cells was performed using the Fisher's exact test. Fisher's exact test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's exact test at any test article dose level, the Cochran-Armitage test was used to measure dose-responsiveness.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of osmolality: The osmolality in treatment medium of the highest concentration tested, 100 µg/mL, was 409 mmol/kg. The osmolality of the solvent (DMSO) in treatment medium was 411 mmol/kg. The osmolality of the test article concentrations in treatment medium are acceptable because they did not exceed the osmolality by more than 20%.
- Precipitation: Visible precipitate was observed in treatment medium at dose levels >/= 420 ug/mL.

RANGE-FINDING/SCREENING STUDIES: CHO cells were exposed to solvent alone and to nine concentrations of test article ranging from 0.14 to 1400 ug/mL in the absence and presence of an S9 reaction mixture. Visible precipitate was observed in treatment medium at dose levels >/= 420 ug/mL. Dose levels < 140 ug/mL were soluble in treatment medium. Substantial toxicity (i.e., at least 50% cell growth inhibition, relative to the solvent control) was observed at dose levels > 140 µg/mL in all three exposure groups

Conclusions:
Under the conditions of this study, the test item did not induce chromosomal aberrations in CHO cells in vitro.
Executive summary:

The test article, 1-Chloro-2,3-dimethylbenzene, was tested in the chromosome aberration assay using Chinese hamster ovary (CHO) cells in both the absence and presence of an Aroclor-induced S9 activation system. A preliminary toxicity test was performed to establish the dose range for the chromosome aberration assay.

Based on the findings of a pre-test, the doses chosen for the chromosome aberration assay ranged from 5 to 100 µg/mL in all experiments. In the chromosome aberration assay, the cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9 activated test system. All cells were harvested 20 hours after treatment initiation. The test article was soluble in treatment medium at all dose levels tested. Selection of doses for microscopic analysis was based on toxicity (the lowest dose with at least 50% reduction in cell growth or mitotic index and two lower doses) in all harvests. The percentage of cells with structural or numerical aberrations in the test article-treated groups was not significantly increased above that of the solvent control at any dose level (p>0.05, Fisher's exact test). Based on the findings of this study, 3-Chloro-o-xylene (CAS # 608-23-1) was concluded to be negative for the induction of structural and numerical chromosome aberrations in CHO cells.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 14 Jul 2003 to 02 Sep 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
February 1998
GLP compliance:
yes
Type of assay:
other: in vitro gene mutation test in mammalian cells (L5178Y/TK+/- Mouse Lymphoma Mutagenesis Assay)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
with and without
Test concentrations with justification for top dose:
Based on the results of a toxicity test, the doses chosen for the mutagenesis assay ranged from 5.0 to 150 µg/mL with and without metabolic activation.
Scored concentrations were:
0, 40, 45, 50, 52, and 54 µg/mL without metabolic activation
0, 50, 60, 75, 100, and 125 µg/mL with metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: based an solubility of the test article and compatibility with the target cells
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h with and without metabolic activation, 24 h without metabolic activation
- Selection time (if incubation with a selection agent): 10-14 days
- Fixation time (start of exposure up to fixation or harvest of cells): up to 15 days

SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF REPLICATIONS: duplicates

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Any supplementary information relevant to cytotoxicity:
Evaluation criteria:
A result was considered positive if a concentration-related increase in mutant frequency was observed and one or more dose levels with 10% or greater total growth exhibited mutant frequencies of >100 mutants per 106 clonable cells over the background level.
A result was considered equivocal if the mutant frequency in treated cultures was between 55 and 99 mutants per 106 clonable cells over the background level.
A result was considered negative if the mutant frequency in treated cultures was fewer than 55 mutants per 106 clonable cells over the background level.
Statistics:
not performed
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Experiment1: 4 h exposure without metabolic activation
The mutant frequencies over vehicle control were (concentration: # in replicate 1; # in replicate 2):
40 µg/ml: -5; 2
45 µg/ml: 29; 23
50 µg/ml: 23; -7
52 µg/ml: 36; 25
54 µg/ml: 74; 52
  
Experiment 2: 4 h exposure with metabolic activation
50 µg/ml: 52;13
60 µg/ml: 3; 1
75 µg/ml: 29; 40
100 µg/ml: 45; -5
150 µg/ml: 9; 22

Experiment 3: 24 exposure without metabolic activation:
45 µg/ml: 35; -4
50 µg/ml: 8; 18
52 µg/ml: -21; -21
54 µg/ml: 12; -11
56 µg/ml: toxic; 13
58 µg/ml: 86; 51
Conclusions:
Under the conditions of this gene mutation study in mammalian cells in vitro, the test item produced an equivocal result without metabolic activation and a negative result with metabolic activation. As the effect in both experiments without metabolic activation was observed only in one of the duplicate culture and without a clear dose-reponse pattern, the findings are considered not to represent a clear indication of a mutagenic response.
Executive summary:

The test article, 1-Chloro-2,3-dimethylbenzene, was tested in the L5178Y/TK+/- Mouse Lymphoma Mutagenesis Assay in the absence and presence of Aroclor-induced rat liver S9.

In the preliminary toxicity assay, the maximum concentration in treatment medium was 1400 µg/ml (10 mM). No visible precipitate was present at any concentration in treatment medium at the end of treatment. Selection of concentrations for the mutation assay was based on reduction of suspension growth relative to the solvent control. Substantial toxicity, i.e., suspension growth of < 50% of the solvent control, was observed at ≥ 150 µg/mL with and without S9 activation with a 4-hour exposure and without activation with a 24-hour exposure.

Based on the results of the preliminary toxicity assay, the doses chosen for treatment of the initial mutagenesis assay ranged from 5.0 to 150 µg/ml for both the non-activated and S9-activated cultures. No visible precipitate was present at any concentration in treatment medium. The concentrations chosen for cloning were 40, 45, 50, 52, and 54 µg/ml without activation and 50, 60, 75, 100, and 125 µg/ml with S9 activation. One non-activated cloned culture exhibited a mutant frequency between 55 and 99 mutants per 10exp6 clonable cells over that of the solvent control. There was no dose-response trend. Toxicity in the cloned cultures, i.e., total growth of < 50% of the solvent control, was observed at doses ≥ 52 µg/ml without activation and ≥ 100 µg/ml with S9 activation.

Based on the results of the preliminary toxicity assay, the doses chosen for treatment of the extended treatment assay ranged from 5.0 to 150 µg/ml for non-activated cultures with a 24-hour exposure. No visible precipitate was present at any concentration in treatment medium. The concentrations chosen for cloning were 45, 50, 52, 54, 56, and 58 µg/ml. One non-activated cloned culture exhibited a mutant frequency between 55 and 99 mutants per 10exp6 clonable cells over that of the solvent control. There was no dose-response trend. Toxicity in the cloned cultures was observed at doses ≥ 56 µg/ml.

The trifluorothymidine-resistant colonies for the positive and solvent control cultures from both assays were sized according to diameter over a range from approximately 0.2 to 1.1 mm. The colony sizing for the MMS positive control yielded the expected increase in small colonies, verifying the adequacy of the methods used to detect small colony mutants. Under the conditions of this study, the test article was concluded to be equivocally mutagenic in mammalian cells without activation with 4- and 24-hour exposures and negative with S9 activation inthe L5178Y/TK+/-Mouse Lymphoma Mutagenesis Assay.

 

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

Genetic toxicity in vivo

Description of key information

The test item did not induce micronuclei in erythrocytes of mice after a single intraperitoneal exposure with doses up to 2000 mg/kg bw (study according OECD guideline 474).

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 21 Jul 2003 to 17 Aug 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
February 1998
GLP compliance:
yes
Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc., Frederick, MD, USA
- Age at study initiation: approx. 6-8 weeks
- Weight at study initiation: 27.0 — 32.3 g (males); 22.1 — 26.3g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: up to 5 of same sex
- Diet: Harlan 2018C Certified Global Rodent Diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 +/- 1.7
- Humidity (%): 50 +/- 20
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: based on a solubility determination of the test article and compatibility of the vehicle with the test system
- Amount of vehicle (if gavage or dermal): dosing volume 20 ml/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: dosing volume 20 ml/kg bw

Duration of treatment / exposure:
1 day
Frequency of treatment:
single exposure
Post exposure period:
24 and 28 h
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 for vehicle controls, 5 in low and mid dose groups and positive control, 15 in high dose group (including 5 replacement animals per sex to ensure the availability of five animals for micronucleus analysis)
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: i.p.
- Doses / concentrations: 50 mg/kg bw
Tissues and cell types examined:
bone marrow of femur
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: based on a pre-test

DETAILS OF SLIDE PREPARATION: The bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 ml fetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended by aspiration with a capillary pipet and a small drop of bone marrow suspension was spread onto a clean glass slide. Two slides were prepared from each mouse. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted

METHOD OF ANALYSIS: Bone marrow cells, polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs) were analyzed for the presence of micronuclei
Evaluation criteria:
the test article was considered to induce a positive response if a dose-responsive increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control at any sampling time. If a single treatment group was significantly elevated at one sacrifice time with no evidence of a dose-response, the assay was considered a suspect or unconfirmed positive and a repeat experiment would have been recommended. The test article was judged negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control values and no evidence of dose response were observed at any sampling time.
Statistics:
Kastenbaum-Bowman Tables
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1, 10, 100, 1000 and 2000 mg/kg bw
- Clinical signs of toxicity in test animals: Clinical signs, which were noted an the days following dose administration, included: lethargy and piloerection at 1000 mg/kg bw and above

RESULTS OF DEFINITIVE STUDY
- Ratio of PCE/NCE (for Micronucleus assay): No appreciable change in the ratio of polychromatic erythrocytes to total erythrocytes relative to the vehicle control was apparent in the test article-treated groups suggesting that the test article did not inhibit erythropoiesis.
- Clinical signs of toxicity in test animals: Clinical signs, which were noted an the days following dose administration, included: lethargy and piloerection at all doses tested
Conclusions:
Under the conditions of this study, the test item did not induce micronuclei in erythrocytes from bone marrow in mice.
Executive summary:

The test article, 1-Chloro-2,3-dimethylbenzene, was investigated in the mouse micronucleus assay. The definitive micronucleus study consisted of seven groups, each containing 5 male and 5 female ICR mice. Animals in five of these groups were treated either with the controls (negative or positive) or with 3-Chloro-o-xylene at a dose of 500, 1000 or 2000 mg/kg bw and were euthanized 24 hours after treatment. Animals in other two groups were treated either with the negative control or test item at 2000 mg/kg and were euthanized 48 hours after treatment. No mortality was observed in any male or female mice in the micronucleus study. Clinical signs, which were noted on the days following dose administration, included: lethargy and piloerection at all doses tested. Bone marrow cells (polychromatic erythrocytes, PCEs and normochromatic erythrocytes, NCEs), collected 24 and 48 hours after, treatment, were examined microscopically for the presence of micronuclei. No appreciable reduction in the ratio of polychromatic erythrocytes to total erythrocytes was observed in the test article-treated groups relative to the vehicle control groups suggesting that the test article did not inhibit erythropoiesis. No significant increase in micronucleated polychromatic erythrocytes in test article-treated groups relative to the respective vehicle control groups was observed in male or female mice at 24 or 48 hours after dose administration.The results of the assay indicate that under the conditions described in this report, a singleintraperitoneal administration of 3-Chloro-o-xylene at doses up to 2000 mg/kg did not induce asignificant increase in the incidence of micronucleated polychromatic erythrocytes in eithermale or female ICR mice. Therefore, 3-Chloro-o-xylene was concluded to be negative in the mouse micronucleus assay.

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

Mode of Action Analysis / Human Relevance Framework

There is no evidence for species specific effects of the substance. Therefore the results of the in vitro and vivo data are regarded as relevant for humans.

Additional information

The test article, 1-Chloro-2,3-dimethylbenzene, was investigated in the Bacterial Reverse Mutation Assay with an Independent Repeat Assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9, using the plate incorporation method. In the initial mutagenicity assay, no positive mutagenic response was observed. The dose levels tested were 0, 15, 50, 150, 500, 1500 and 5000 μg/plate with tester strain WP2 uvrA in the presence of S9 activation and 0, 5.0, 15, 50, 150, 500 and 1500 μg/plate with all other test conditions. Toxicity was observed beginning at 150, 500, 1500 or 5000 in per plate. No precipitate was observed. In the independent repeat mutagenicity assay, no positive mutagenic response was observed. The dose levels tested were 0, 7.5, 25, 75, 200, 600, 1800 and 5000 μg/plate with tester strains TA98 and WP2 uvrA in the presence of S9 activation and 2.5, 7.5, 25, 75, 200 and 600 μg/plate with all other test conditions. Toxicity was observed beginning at 75, 200, 600, 1800 or 5000 μg/plate. No precipitate was observed. Under the conditions of this study, test article 3-Chloro-o-xylene was concluded to be negative in the Bacterial Reverse Mutation Assay with an Independent Repeat Assay.

The test article, 1-Chloro-2,3-dimethylbenzene, was investigated in the chromosome aberration assay using Chinese hamster ovary (CHO) cells in both the absence and presence of an Aroclor-induced S9 activation system. A preliminary toxicity test was performed to establish the dose range for the chromosome aberration assay. Based on the findings of a pre-test, the doses chosen for the chromosome aberration assay ranged from 5 to 100 μg/mL in all experiments. In the chromosome aberration assay, the cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9 activated test system. All cells were harvested 20 hours after treatment initiation. The test article was soluble in treatment medium at all dose levels tested. Selection of doses for microscopic analysis was based on toxicity (the lowest dose with at least 50% reduction in cell growth or mitotic index and two lower doses) in all harvests. The percentage of cells with structural or numerical aberrations in the test article-treated groups was not significantly increased above that of the solvent control at any dose level. Based on the findings of this study, 3-Chloro-o-xylene (CAS # 608-23-1) was concluded to be negative for the induction of structural and numerical chromosome aberrations in CHO cells.

The test article, 1-Chloro-2,3-dimethylbenzene, was investigated in the L5178Y/TK+/- Mouse Lymphoma Mutagenesis Assay in the absence and presence of Aroclor-induced rat liver S9. Based on the results of a preliminary toxicity assay, the doses chosen for treatment ranged from 5.0 to 150 μg/ml for both the non-activated and S9-activated cultures for the 4 h exposure. No visible precipitate was present at any concentration in treatment medium. The concentrations chosen for cloning were 40, 45, 50, 52, and 54 μg/ml without activation and 50, 60, 75, 100, and 125 μg/ml with S9 activation. One non-activated cloned culture exhibited a mutant frequency between 55 and 99 mutants per 10exp6 clonable cells over that of the solvent control. There was no dose-response trend. Toxicity in the cloned cultures, i.e., total growth of < 50% of the solvent control, was observed at doses ≥ 52 μg/ml without activation and ≥ 100 μg/ml with S9 activation. The doses chosen for treatment of the extended treatment assay ranged from 5.0 to 150 μg/ml for non-activated cultures with a 24-hour exposure. No visible precipitate was present at any concentration in treatment medium. The concentrations chosen for cloning were 45, 50, 52, 54, 56, and 58 μg/ml. One non-activated cloned culture exhibited a mutant frequency between 55 and 99 mutants per 10exp6 clonable cells over that of the solvent control. There was no dose-response trend. Toxicity in the cloned cultures was observed at doses ≥ 56 μg/ml. Under the conditions of this study, the test article was concluded to be equivocally mutagenic without activation with 4- and 24-hour exposures and negative with S9 activation inthe L5178Y/TK+/-Mouse Lymphoma Mutagenesis Assay. As the effect in both experiments without metabolic activation was observed only in one of the duplicate culture and without a clear dose-reponse pattern, the findings are considered not to represent a clear indication of a mutagenic response.

The test article, 1-Chloro-2,3-dimethylbenzene, was investigated in the mouse micronucleus assay at doses of 500, 1000 or 2000 mg/kg bw. No mortality was observed in any male or female mice in the micronucleus study. Clinical signs, which were noted on the days following dose administration, included: lethargy and piloerection at all doses tested. Bone marrow cells (polychromatic erythrocytes, PCEs and normochromatic erythrocytes, NCEs), collected 24 and 48 hours after treatment, were examined microscopically for the presence of micronuclei. No appreciable reduction in the ratio of polychromatic erythrocytes to total erythrocytes was observed in the test article-treated groups relative to the vehicle control groups suggesting that the test article did not inhibit erythropoiesis. No significant increase in micronucleated polychromatic erythrocytes in test article-treated groups relative to the respective vehicle control groups was observed in male or female mice at 24 or 48 hours after dose administration.Therefore, the test article was concluded to be negative in themouse micronucleus assay.

Justification for classification or non-classification

Due to the predominantly negative findings in in vitro assays and the negative results of the in vivo study no classification for mutagenicity is required according to the criteria of Regulation (EC) No 1272/2008.