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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test:

Test chemical did not induce mutation in Salmonella Typhimurium TA 1538, 1535, 1537, 98 and 100 with and without mammalian S-9 microsomal fraction. Hence it is not likely to classify as a gene mutant in vitro.

Chromosome aberration: The test chemical did not induce chromosome aberration in the CHL/IU cells both in the presence and absence of S9 activation system and hence it is not likely to be gene mutant in vitro.

Mammalian cell Gene mutation toxicity:

Test chemical did not induce mutation in L5178Y TK ± mouse lymphoma with and without mammalian S-9 microsomal fraction. Hence it is not likely to classify as a gene mutant in vitro.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Justification for type of information:
Data is fom secondary source
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
In – vitro Bacterial reverse mutation assay of test chemical in S. typhimurium.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
not specified
Species / strain / cell type:
S. typhimurium, other: TA 1538, 1535, 1537, 98 and 100
Details on mammalian cell type (if applicable):
not specified
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 activation
Test concentrations with justification for top dose:
1, 4, 16, 64 and 256 mg/plate (without S-9 activation)4, 16, 64, 256 and 1024 mg/plate (with S-9 activation)
Vehicle / solvent:
Double deionized water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Double deionized water
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Remarks:
not specified
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
NUMBER OF REPLICATIONS: Three plates per dose level
Rationale for test conditions:
not specified
Evaluation criteria:
reverse mutations
Statistics:
not specified
Species / strain:
S. typhimurium, other: TA 1538, 1535, 1537, 98 and 100
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not specified
Additional information on results:
In the initial toxicity assays, the test substance without metabolic activation was cytotoxic to the tester strains TA 1535 and TA 1537 at 1000 mg/plate and cytotoxic to tester strain TA 1538 at 200 mg/plate. The test substance with metabolic activation was cytotoxic to the tester strains TA 1535 and TA 1538 at 5000 mg/plate and tester strain TA 1537 at 1000 mg/plate.
Remarks on result:
other: Non mutagenic
Conclusions:
Test chemical did not induce mutation in Salmonella Typhimurium TA 1538, 1535, 1537, 98 and 100 both in the presence and absence of mammalian S-9 microsomal fraction and hence it is not likely to classify as a gene mutant in vitro.
Executive summary:

Gene toxicity test was performed to determine the mutagenic nature of test chemical. The study was performed using Salmonella Typhimurium TA 1538, 1535, 1537, 98 and 100 both in the presence and absence of mammalian S-9 microsomal fraction. The test material did not induce reverse mutations. Therefore, Test chemical did not induce mutation in Salmonella Typhimurium TA 1538, 1535, 1537, 98 and 100 with and without mammalian S-9 microsomal fraction. Hence it is not likely to classify as a gene mutant in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from publication
Qualifier:
according to
Version / remarks:
as below
Principles of method if other than guideline:
Gene toxicity study of test chemical in L5178Y TK ±mouse lymphoma assay.
GLP compliance:
not specified
Type of assay:
other: mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
not specified
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
not specified
Metabolic activation:
with and without
Metabolic activation system:
not specified
Test concentrations with justification for top dose:
0.001, 0.01, 0.1, 1.0, 10 and 100 µL/mL
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Remarks:
not specified
Details on test system and experimental conditions:
not specified
Rationale for test conditions:
not specified
Evaluation criteria:
Mutation frequency were observed
Statistics:
not specified
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
Not specified
Remarks on result:
other: No mutagenic potential observed
Conclusions:
Test chemical did not induce mutation in L5178Y TK ± mouse lymphoma and hence it is not likely to classify as a gene mutant in vitro.
Executive summary:

Gene toxicity test was performed to determine the mutagenic nature of test chemical. The study was performed using L5178Y TK ± mouse lymphoma both in the presence and absence of mammalian S-9 microsomal fraction. The test material did not significantly increase mutation frequency over the average mutation frequency of solvent control. Therefore, Test chemical did not induce mutation in L5178Y TK ± mouse lymphoma with and without mammalian S-9 microsomal fraction. Hence it is not likely to classify as a gene mutant in vitro.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames assay:

Data available for the target chemical was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

Study 1: Gene toxicity test was performed to determine the mutagenic nature of test chemical. The study was performed using Salmonella Typhimurium TA 1538, 1535, 1537, 98 and 100 both in the presence and absence of mammalian S-9 microsomal fraction. The test material did not induce reverse mutations. Therefore, Test chemical did not induce mutation in Salmonella Typhimurium TA 1538, 1535, 1537, 98 and 100 with and without mammalian S-9 microsomal fraction. Hence it is not likely to classify as a gene mutant in vitro.

Study 2: Gene toxicity test was performed to determine the mutagenic nature of test chemical. The study was performed using Salmonella Typhimurium other both in the presence and absence of mammalian S-9 microsomal fraction. The test material did not induce reverse mutations. Therefore, Test chemical did not induce mutation in Salmonella Typhimurium other with and without mammalian S-9 microsomal fraction. Hence it is not likely to classify as a gene mutant in vitro.

Study 3: Gene toxicity test was performed to determine the mutagenic nature of test chemical. The study was performed using S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 both in the presence and absence of mammalian S-9 microsomal fraction. The test material did not induce reverse mutations. Therefore, Test chemical did not induce mutation in Salmonella Typhimurium TA 1535, TA 1537, TA 98 and TA 100 with and without mammalian S-9 microsomal fraction. Hence it is not likely to classify as a gene mutant in vitro.

Study 4:Gene toxicity test was performed to determine the mutagenic nature of test chemical. The study was performed using S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 both in the presence and absence of mammalian S-9 microsomal fraction. The test material caused a visible reduction in the growth of the bacterial lawn to all of the strains tested. The first evidence of toxicity was observed at 150 mg/plate. No significant increase in the frequency of revertant colonies of bacteria was recorded for any of the strains used, at any dose level with or without metabolic activation. All of the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies and the activity of the S9 fraction was found to be satisfactory. The test material did not induce reverse mutations. Therefore, Test chemical did not induce mutation in Salmonella Typhimurium TA 1535, TA 1537, TA 98 and TA 100 with and without mammalian S-9 microsomal fraction. Hence it is not likely to classify as a gene mutant in vitro.

  

Study 5:Gene mutation study was performed on Salmonella Typhimurium strains to determine the mutagenic nature of test chemical. Salmonella Typhimurium TA 1538, 1535, 1537, 98, 100 and E. coli WP2 were used during the study. The study was performed in the presence and absence of S9 metabolic activation. The S9 fraction was prepared from the liver of male Sprague-Dawley rats, weighing about 220g (seven weeks old), pretreated with polychlorinated biphenyl (KC 500) at a dose of 500mg/kg body weight five days before sacrifice. The positive controls used were 4-nitroquinoline-N-oxide, 9-aminoacridine, 2-nitrofluorene, N-ethyl-N-nitro-N-nitrosoguanidine, benzo(a)pyrene, 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2) and 9-aminoacridine. 0.1ml of bacterial strain and 0.5ml of S9 mix or sodium phosphate buffer (pH 7.4) were added to a sterile test tube containing 0.1ml of various concentrations of the chemical. The test concentrations used were 1, 5, 10, 50, 100, 500, 1000 and 5000 µg/plate and the test chemical was dissolved in DMSO. This mixture was preincubated in a shaker water bath at 37°C for 20 minutes, and then added to 2ml of molten top agar (45°C). This mixture was preincubated in a shaker water bath at 37°C for 20 minutes, and then added to 2ml of molten top agar (45°C). The contents of each tube were mixed and poured onto a minimal glucose agar plate immediately. The plates were incubated at 37°C for 48 hours, and then the number of revertant colonies on each plate was scored with an automated colony counter. The background bacterial lawn was checked routinely by dissected microscope. Negleble amount of revertant colonies were observed as compared to positive control substances. Thus the test chemical can be considered as non-mutagenic when Salmonella Typhimurium TA 1538, 1535, 1537, 98, 100 and E. coli WP2 were treated in the presence and absence of S9 activation system.

Test chemical did not induce gene mutation in Salmonella Typhimurium strains TA 1538, 1535, 1537, 98, 100 and E. coli WP2 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

 

 

Cromosomal Abberation:

 

Data available for the target chemical was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

 

In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster V79 cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in suitable solvent and used. The doses for the main study were based on the preliminary dose range finding study. Evaluated dose levels were 1.0 μg/ml without S9-mix and 10.0 μg/ml with S9-mix for 7 hours; 0.3, 1.0 and 3.0 μg/ml without S9-mix and 1.0, 3.0 and 10.0 μg/ml with S9-mix for 18 hours; and 3.0 μg/ml without S9-mix and 10.0 μg/ml with S9-mix for 28 hours.Logarithmically growing cells were incubated with the test substance in serum-free culture medium for 4 hrs. Cells were subsequently washed in glucose-containing saline and cultured in normal medium for 7, 18 and 28 hours. Ethylmethanesulfonate and cyclophosphamide were used as positive controls in 18-hour cultures without and with S9-mix, respectively. Two hours (7 hour interval) or 2.5 hours (18 and 28 hours intervals) before the end of the incubation period, colcemid was added to the cultures. The cells were put onto glass slides, treated with hypotonic potassium chloride solution, fixed in methanol and acetic acid and stained with Giemsa solution. In each experimental group two parallel cultures were set up. Per culture 100 metaphases were scored for structural chromosomal aberrations (breaks, fragments, deletions, exchanges and chromosomal disintegrations). Chromosomal gaps were recorded separately. There were no biologically relevant and statistically significant increases in cells with structural aberrations after treatment with the test substance at any fixation interval either with or without metabolic activation. Based on the observations made, the test chemical did not induce chromosome aberration in the Chinese hamster V79 cells both in the presence and absence of S9 activation system and hence it is not likely to be gene mutant in vitro.

 

In another experiment, vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using CHL/IU cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in physiological saline and used at dose level of 0, 100, 125, 150, 175 or 200 µg/mL (short term treatment method) and 0, 50, 75, 100, 125 or 150 µg/mL (main test) and 0, 100, 110, 120, 130, 140 or 150 µg/mL (additional test) without S9 in the continuous treatment method. The doses for the main study were based on the preliminary dose range finding study. The test was performed by continuous treatment method for 24 hrs and short term treatment method for 6 hrs. The cells were allowed to express for 18 hrs in short term treatment method. Two hours before the end of the culture, colcemid was added to the culture solution to a final concentration of about 0.1 μg / ml. Chromosome specimens were prepared according to a conventional method. Two slide specimens were prepared for each dish. The prepared specimen was stained with 3% Giemsa solution for 20 mins. Chromosomes were analyzed based on the classification method by the Japan Society for Environmental Mutagenesis and Mammalian Testing, and structural abnormalities such as breakage and exchange of chromosome type or chromosome type, presence and absence of gap, and ploidy. The presence or absence of cells (polyploid) was observed. The gap was not included in structural abnormality. The frequency of cells with chromosomal abnormality is negative, less than 5% negative, less than 10% false positive, more than 10% Positive. Based on the observations made, the test chemical did not induce chromosome aberration in the CHL/IU cells both in the presence and absence of S9 activation system and hence it is not likely to be gene mutant in vitro.

 

Test chemical did not induce chromosomal aberration in Chinese hamster V79 cells and CHL/IU cells in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

 

in vitro gene mutation study in mammalian cells:

Data available for the target chemical was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

 

Gene toxicity test was performed to determine the mutagenic nature of test chemical. The study was performed using L5178Y TK ± mouse lymphoma both in the presence and absence of mammalian S-9 microsomal fraction. The test material did not significantly increase mutation frequency over the average mutation frequency of solvent control. Therefore, Test chemical did not induce mutation in L5178Y TK ± mouse lymphoma with and without mammalian S-9 microsomal fraction. Hence it is not likely to classify as a gene mutant in vitro.

 

The test chemical was tested in the L5178Y TK+/- Mouse Lymphoma Mutagenesis assay in the presence and absence of rat liver S-9 to determine the mutagenic nature of the test chemical. The study was performed at various concentrations 0.0038 to 0.050 μl/ml (10 concentrations) without S9 and 0.012 to 0.16 μl/ml (10 concentrations) with S9 metabolic activation system. The cells were exposed to the test chemical, positive control and negative control for 4 hours. An expression time of 2 days was allowed with cell population adjustment at 24 and 48 hours. At the end of the expression period, the cells were placed in cloning medium. Cell counts were made for each preparation and the appropriate number of cells were removed and plated. Total number of colonies per plate and the mutation frequency were determined. None of the treated cultures that were cloned exhibited a significant increase in mutant frequency over the average mutant frequency of the solvent controls and no dose response was observed. Thus, the test chemical was considered to be not mutagenic in nature.

Test chemical did not induce mutation in L5178Y TK ± mouse lymphoma with and without mammalian S-9 microsomal fraction. Hence it is not likely to classify as a gene mutant in vitro.

Justification for classification or non-classification

Based on the data available for test chemical, it does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.