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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test material was non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

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:
17.10.2001-03.04.2002
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:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
HIS operon (S. thyphimurium)TRY operon (E. coli)
Species / strain / cell type:
S. typhimurium TA 1535
Details on mammalian cell type (if applicable):
his G 46, 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
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 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 102
Details on mammalian cell type (if applicable):
his G 428, rfa + R-factor
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: mutations in the tryptophan operon
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate (S9 mix) from Aroclor 1254-pretreated rats with standard co-factors
Test concentrations with justification for top dose:
The test material should be investigated in the first experimental series at seven concentrations, separated by half-log intervals (i.e. a factor of sqrt(10)), usually ranging from 5.00 to 5000 μg per plate. Due to limited solubility of the test material in the solvent used for this investigation, the maximum concentration of 2810 μg per plated could be tested. In the second experimental series, usually 5 concentrations including at least 4 non-toxic concentrations should be tested. Signs of toxicity to the bacteria are a reduction in the number of spontaneous revertants or a clearing of background lawn of nonrevertant bacteria. Precipitation of the test material, if it occurs, should not interfere with scoring of the colonies. Depending upon the precipitation characteristics of the
test material and toxicity data ascertained in the first series, high concentrations may be omitted or lower concentrations may additionally be tested in the second experimental series.
On the basis of the criteria stated above, the following test material concentrations were used for the test item:
1st series: 2.81, 8.89, 28.1, 88.9, 281, 889 and 2810 μg per plate
2nd series: 2.81, 8.89, 28.1, 88.9 and 281 μg per plate
Vehicle / solvent:
DMSO (> 99 %)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
cumene hydroperoxide
other: without S9: daunomycin; with S9: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 3-5 hours
- Exposure duration: about 2 days

SELECTION AGENT (mutation assays): Histidine, Tryptophane

NUMBER OF REPLICATIONS:
Negative controls: 6
Test material : 3
Positive controls: 3

NUMBER OF CELLS EVALUATED: about 1e9

DETERMINATION OF CYTOTOXICITY
- Method: background cytotoxicity
Rationale for test conditions:
According to guideline
Evaluation criteria:
- valid assay (cf. laboratory historical data)
- no or weak increase in revertant colonies = negative
- clear, dose dependent (over at least two concentrations), and reproducible increase in revertant colonies= positve
Statistics:
No statistical evaluation of the data is required.
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:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
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:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
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:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
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:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Water solubility: ok
- Precipitation: yes (≥ 50 µg/plate)
- Other confounding effects: no
Conclusions:
The test material was non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

The investigations for mutagenic potential were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA in a study conducted according to OECD 471. 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. In the two series with S9 mix, 10 % and 30 % S9 in the S9 mix were used in the 1st and 2nd series, respectively


The test material was dissolved in acetone and tested at concentrations ranging from 2.81 to 2810 µg/plate. Precipitation of the test material on the agar plates occurred at concentrations ≥ 50 µg/plate. Toxicity to the bacteria was not observed.


Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine and cumene hydroperoxide served as strain specific positive control test materials in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. 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 performed, there were no treatment related increasing in the mutation frequencies observed with and without metabolic activation.


With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described. The test material was non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The investigations for mutagenic potential were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA in a study conducted according to OECD 471. 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. In the two series with S9 mix, 10 % and 30 % S9 in the S9 mix were used in the 1st and 2nd series, respectively


The test material was dissolved in acetone and tested at concentrations ranging from 2.81 to 2810 µg/plate. Precipitation of the test material on the agar plates occurred at concentrations ≥ 50 µg/plate. Toxicity to the bacteria was not observed.


Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine and cumene hydroperoxide served as strain specific positive control test materials in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. 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 performed, there were no treatment related increasing in the mutation frequencies observed with and without metabolic activation.


With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described. The test material was non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008


The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity in vitro, the test item doesnot require classification according to Regulation (EC) No 1272/2008 (CLP).