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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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

Data source

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

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Density: 1.05 g/cm3

Method

Species / strainopen allclose all
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
Controlsopen allclose all
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

Results and discussion

Test resultsopen allclose all
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.

Applicant's summary and conclusion

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.