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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471, RL1): negative with and without metabolic activation

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24.04.-02.05.1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
HIS operon (S. thyphimurium)
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
his G 46, uvrB, rfa + R-factor
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 1535
Details on mammalian cell type (if applicable):
his G 46, uvrs, rfa
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
his D 3052, uvrB, rfa + R-factor
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
his D 3052, uvrB, rfa
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 1537
Details on mammalian cell type (if applicable):
his C 3076, uvrB, rfa
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate (S9 mix) with standard co-factors with metabolic activation (Aroclor)
Test concentrations with justification for top dose:
The test material concentrations were used selected according to the EC and OECD guidelines for this test system :
50, 250, 1250, 2500, 5000, and 10000 μg/plate.
Vehicle / solvent:
Acetone
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, 9-aminoacridine, daunomycin, 1-ethyl-2-nitro-3-nitrosoguanidine, methyl methanesulfonate, 2-nitrofluorene, 4-nitro-1,2-phenylene diamine, sodium azide
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : four
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:

- Exposure duration/duration of treatment: 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants; a clearing of the background lawn; reduced survival

METHODS FOR MEASUREMENTS OF GENOTOXICIY: Revertant colonies were scored using a colony counter or manually.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
True 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:
no cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
True 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:
no cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
True 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:
no cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid

Results

The test item was examined for mutagenic activity in two series of in vitro microbial assays employing Salmonella typhimurium as indicator organism. The test material was tested directly and after metabolic activation by liver S-9 fraction obtained from rats treated with Aroclor for induction of microsomal enzymes.

 

The data are presented on pages 17 (Range finding) and 18-27 (Mutagenicity tests) in the attached background material.

 

The number of revertants is given per plate and as mean value ± standard deviation.

 

The results of both series of experiments, each performed with and without the addition of rat liver microsomal fraction as external metabolizing system, were negative.

 

Under the test conditions used, the test item was not mutagenic in Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, and TA 1538. In this study toxic effects were not observed as anticipated from the results of the range finding test.

 

The validity of the mutation assay can be assessed by the results obtained for the positive and negative controls. The negative control mutant frequencies were all in the regular range, and the positive control compounds yielded the expected mutant frequencies that were greatly in excess of the background.

Conclusions:
With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.
Executive summary:

Purpose

The purpose of this assay was to provide information on possible health hazards for the test material and serve as a rational basis for risk assessment to the genotoxic potential of the test item in man.

Study Design

The investigations for the mutagenic potential of the test material were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537, and TA 1538. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed.

Results

The test material was dissolved in Acetone and tested at concentrations ranging from 50 to 10000 µg/plate. No evidence for toxicity to the bacteria has been obtained.

2-aminoanthracene, 9-aminoacridine, daunomycin, 1-ethyl-2-nitro-3-nitrosoguanidine, methyl methanesulfonate, 2-nitrofluorene, 4-nitro-1,2-phenylene diamine, sodium azide served as strain specific positive control test materials. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

In both series of experiments, each performed with and without the addition of rat liver S9 mix as the external metabolizing system, the test material showed no increase in the number of revertants of any bacterial strain. Therefore, the test material was not mutagenic under the described experimental conditions.

Conclusion

With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial Reverse Mutation Assay (OECD 471)

The test material was dissolved in Acetone and tested at concentrations ranging from 50 to 10000 µg/plate. No evidence for toxicity to the bacteria has been obtained.

2-aminoanthracene, 9-aminoacridine, daunomycin, 1-ethyl-2-nitro-3-nitrosoguanidine, methyl methanesulfonate, 2-nitrofluorene, 4-nitro-1,2-phenylene diamine, sodium azide served as strain specific positive control test materials. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

In both series of experiments, each performed with and without the addition of rat liver S9 mix as the external metabolizing system, the test material showed no increase in the number of revertants of any bacterial strain.

With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.

Justification for classification or non-classification

Based on the available data, there is no indication for a mutagenic in vitro. The available data on genetic toxicity are conclusive but not sufficient to meet the criteria for classification according to Regulation (EC) 1272/2008.