Registration Dossier
Registration Dossier
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EC number: 231-335-2 | CAS number: 7493-74-5
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Ames test (OECD 471): negative in S.
typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coil WP2 uvrA, with
and without metabolic activation
HPRT (OECD 476): negative in V79 cells with and without metabolic activation
In vitro mammalian cell micronucleus test (OECD 487): negative in cultured human lymphocytes with and without metabolic activation
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 29 July - 04 Sep 2015
- 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)
- Version / remarks:
- 2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon (for S. typhimurium strains)
trp operon (for E. coli strain) - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone
- Test concentrations with justification for top dose:
- Pre-Experiment
3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation.
The pre-experiment is reported as Experiment I.
Experiment II
without metabolic activation:
1, 3, 10, 33, 100, 333, 1000 and 2500 µg/plate (TA1535, TA1537, TA98 and TA100)
33, 100, 333, 1000, 2500 and 5000 µg/plate (WP2 uvrA)
with metabolic activation:
3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate (TA1535, TA1537, TA98 and TA100)
33, 100, 333, 1000, 2500 and 5000 µg/plate (WP2 uvrA) - Vehicle / solvent:
- - Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria. - Untreated negative controls:
- yes
- Remarks:
- untreated controls
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- methylmethanesulfonate
- other: 2-aminoanthracene (2-AA); 4-nitro-o-phenylene-diamine (4-NOPD)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation) (Experiment I); preincubation (Experiment II)
DURATION
- Preincubation period: 1 h
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn - Evaluation criteria:
- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant. - Statistics:
- Mean values and standard deviation were calculated.
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- in all Salmonella strains with and without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item was observed in the overlay agar in the test tubes. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate in the presence of S9 mix in both experiments. The undissolved particles had no influence on the data recording.
RANGE-FINDING/SCREENING STUDIES: The pre-experiment is reported as Experiment I.
ADDITIONAL INFORMATION ON CYTOTOXICITY: The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. Only in Experiment II reduced background growth was observed in the absence of S9 mix at 2500 µg/plate in strain TA 100 and at 5000 µg/plate in strain WP2 uvrA. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all Salmonella strains with and without metabolic activation. Please refer to table 3. - Conclusions:
- Under the conditions of the Ames test the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA) tested with and without metabolic activation.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 11 Aug - 27 Oct 2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Version / remarks:
- 1998
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
- Type of assay:
- other: mammalian cell gene mutation assay
- Target gene:
- HPRT locus
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MEM containing Hank's salts, 10% FBS (except during 4 h treatment), neomycin (5 µg/mL) and amphotericin B (1%)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone
- Test concentrations with justification for top dose:
- Pre-Experiment:
4 h and 24 h treatment: 15, 30.1, 60.1, 120.3, 240.5, 481, 962 and 1924 µg/mL without metabolic activation
4 h treatment: 15, 30.1, 60.1, 120.3, 240.5, 481, 962 and 1924 µg/mL with metabolic activation
Experiment 1:
4 h treatment: 30, 60, 120, 240, 480, 720 and 960 µg/mL without metabolic activation
4 h treatment: 15, 30, 60, 120, 240, 360 and 480 µg/mL with metabolic activation
Experiment 2:
24 h treatment: 30, 60, 120, 240, 480, 720 and 960 µg/mL without metabolic activation
4 h treatment: 15, 30, 60, 120, 180, 240, 300 and 360 µg/mL with metabolic activation - Vehicle / solvent:
- - Vehicle/solvent used: DMSO (0.5% (v/v))
- Justification for choice of solvent/vehicle: The solvent was chosen based on its solubility properties and its relative non-toxicity to the cell cultures. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
1st experiment: 4 h exposure with and without S9 mix
2nd experiment: 4 h exposure with S9 mix and 24 h without S9 mix
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days
SELECTION AGENT (mutation assays): 11 µg/mL 6-thioguanine (6-TG)
NUMBER OF REPLICATIONS: duplicates each in two independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: relative cloning efficiency I or cell density below 50% - Evaluation criteria:
- A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations of the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration. - Statistics:
- A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 480 µg/mL and above without metabolic activation and at 360 µg/mL with metabolic activation in Exp. I and at 180 µg/mL with metabolic activation in Exp. II
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolarity: There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
- Precipitation: The test medium was checked for precipitation or phase separation at the end of each treatment period (4 h) prior to removal to the test item.
Pre-Experiment:
Phase separation occurred at 481 µg/mL and above after 4 h treatment without metabolic activation and at 240.5 µg/mL and above with metabolic activation. Following 24 h treatment phase separation was observed at 240.5 µg/mL and above.
Main Experiment:
Phase separation was observed after 4 h treatment at 480 µg/mL and above in Experiment I without metabolic activation and at 360 µg/mL and above with metabolic activation. In the second experiment phase separation occurred at 480 µg/mL and above without metabolic activation and at 240 µg/mL and above with metabolic activation.
RANGE-FINDING/SCREENING STUDIES:
A pre-experiment was performed in order to determine the concentration range for the mutagenicity experiments. The pre-experiment was performed in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Test item concentrations between 15 µg/mL and 1924 µg/mL (equal to a molar concentration of approx. 10 mM) were used. The highest concentration of the pre-experiment was chosen with regard to the purity (99.1%) and the molecular weight (192.23 g/mol) of the test item. Strong cytotoxic effects occurred after 4 h treatment at 1924 µg/mL without and at 240.5 µg/mL and above with metabolic activation. Following 24 h treatment, severe cytotoxicity was noted at 962 µg/mL and above.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
The cultures at the highest concentration with and without metabolic activation were not continued based on exceedingly severe cytotoxic effects in Experiment I. In Experiment II the cultures at the two highest concentrations without and the three highest concentrations with metabolic activation were not continued due to exceedingly severe cytotoxic effects.
The recommended toxic range of approx. 10-20% cloning efficiency I was covered in the insoluble range with metabolic activation. In the insoluble range of the experimental parts without metabolic activation cytotoxicity was either below or above the recommended range but visible phase separation occurred. - Conclusions:
- Under the experimental conditions of the gene mutation assay the test item did not induce gene mutations at the HPRT locus in V79 cells with and without metabolic activation.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 23 Sep - 27 Oct 2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test) 2014
- Version / remarks:
- adopted 26 Sep 2014
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
- Type of assay:
- in vitro mammalian cell micronucleus test
- Target gene:
- not applicable
- Species / strain / cell type:
- lymphocytes: cultured peripheral human lymphocytes
- Details on mammalian cell type (if applicable):
- - Cell proliferation: Blood was collected from healthy non-smoking donors not receiving medication. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes. Human lymphocytes were stimulated for proliferation by the addition of the mitogen phytohemeagglutinine (PHA) to the culture medium for a period of 48 h. The cell harvest time point was approx. 2 – 2.5 x AGT (average generation time).
- Type and identity of media: DMEM/F12, mixture 1:1 already supplemented with 200 mM GlutaMAX. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 µg/mL), the mitogen PHA (3 µg/mL), 10% FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL)
- Properly maintained: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone
- Test concentrations with justification for top dose:
- Pre-Experiment / Experiment I:
4 h treatment: 12.5, 21.8, 38.2, 66.9, 117.1, 204.9, 358.6, 627.6, 1098.3 and 1922 µg/mL with and without metabolic activation
Since the cultures fulfilled the requirements for cytogenetic evaluation, the preliminary test was designated Experiment I
Experiment 2:
20 h treatment: 38.2, 66.9, 117.1, 204.9, 358.6, 627.6, 1098.3 and 1922 µg/mL without metabolic activation - Vehicle / solvent:
- - Vehicle/solvent used: DMSO (0.5% (v/v)
- Justification for choice of solvent/vehicle: The solvent was chosen based on its solubility properties and its relative non-toxicity to the cell cultures. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: demecolcin
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 and 20 h
- Fixation time (start of exposure up to fixation or harvest of cells): 4 h treatment: 40 h; 20 h treatment: 40 h
ACTIN POLYMERISATION INHIBITOR (cytogenetic assays): cytochalasin B, 4 µg/mL
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 1000 binucleated cells per culture
DETERMINATION OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index (CBPI) - Evaluation criteria:
- The micronucleus assay will be considered acceptable if it meets the following criteria:
− The concurrent solvent control will normally be within the laboratory historical solvent control data range.
− The concurrent positive controls should induce responses that are compatible with the laboratory historical positive control data and produce a statistically significant increase.
− Cell proliferation criteria in the solvent control are considered to be acceptable.
− All experimental conditions were tested unless one exposure condition resulted in a clearly positive result.
− The quality of the slides must allow the evaluation of an adequate number of cells and concentrations.
A test substance is considered to be clearly negative if, in all of the experimental conditions examined:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data
A test substance is considered to be clearly positive if, in any of the experimental conditions examined:
− At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition
− The results are outside the range of the laboratory historical solvent control data - Statistics:
- Chi square test (p < 0.05)
- Species / strain:
- lymphocytes: cultured peripheral human lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolarity: No relevant influence on osmolarity or pH value was observed.
- Precipitation: No precipitation of the test substance in the culture medium was observed. In Experiment I and II phase separation of the test substance in the culture medium was observed at 627.6 µg/mL and above in the absence and presence of S9 mix.
RANGE-FINDING/SCREENING STUDIES:
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Test substance concentrations ranging from 12.5 to 1922 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-experiment for toxicity, phase separation of the test substance was observed at the end of treatment at 627.6 µg/mL and above in the absence and presence of S9-mix. Since the cultures fulfilled the requirements for cytogenetic evaluation, the preliminary test was designated Experiment I
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. - Conclusions:
- Under the experimental conditions of the in vitro micronucleus test the test substance did not induce micronuclei in human lymphocytes with and without metabolic activation.
Referenceopen allclose all
Table 1. Test results of main test 1 (plate incorporation).
With or without S9-Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation) |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA1535 |
TA100 |
WP2 uvrA |
TA1537 |
TA98 |
||
- |
0 (DMSO) |
9 ± 1 |
138± 8 |
44 ± 2 |
9 ± 2 |
28± 7 |
- |
0 |
11 ± 2 |
164± 6 |
38 ± 5 |
14 ± 6 |
25± 4 |
- |
3 |
11 ± 4 |
150± 7 |
52 ± 3 |
9 ± 3 |
22± 3 |
- |
10 |
10 ± 4 |
134± 12 |
38 ± 10 |
11 ± 4 |
20 ± 2 |
- |
33 |
9 ± 3 |
156± 21 |
45 ± 8 |
10 ± 2 |
26± 8 |
- |
100 |
8 ± 2 |
151± 10 |
44 ± 9 |
11 ± 4 |
21± 8 |
- |
333 |
6 ± 1 |
126± 11 |
43 ± 2 |
6 ± 1 |
20± 3 |
- |
1000 |
3 ± 1 |
43± 11 |
38 ± 7 |
2 ± 1 |
10± 4 |
- |
2500 |
1 ± 1 |
28± 10 |
43 ± 4 |
2 ± 1 |
7± 0 |
- |
5000 |
0 ± 0 |
7± 2 |
29 ± 2 |
0 ± 0 |
4± 2 |
Positive controls, –S9 |
Name |
NaN3 |
NaN3 |
MMS |
4-NOPD |
4-NOPD |
Concentrations (μg/plate) |
10 |
10 |
2.0 µL |
50 |
10 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
1288 ± 46 |
2161± 203 |
926 ± 110 |
81 ± 8 |
299± 19 |
|
+ |
0 (DMSO) |
13 ± 6 |
112± 17 |
43 ± 9 |
8 ± 2 |
26± 4 |
+ |
0 |
12 ± 3 |
151± 8 |
54 ± 4 |
10 ± 3 |
32± 11 |
+ |
3 |
12 ± 5 |
109± 16 |
43 ± 7 |
10 ± 3 |
32± 3 |
+ |
10 |
12 ± 4 |
100± 16 |
44 ± 2 |
9 ± 4 |
25± 3 |
+ |
33 |
6 ± 2 |
117± 7 |
47 ± 12 |
8 ± 2 |
25± 8 |
+ |
100 |
7 ± 1 |
115± 8 |
63 ± 8 |
7 ± 1 |
32± 4 |
+ |
333 |
7 ± 2 |
115± 6 |
58 ± 4 |
9 ± 2 |
27± 6 |
+ |
1000 |
4 ± 1 |
94± 13 |
51 ±19 |
7 ± 1 |
14± 2 |
+ |
2500 |
0 ± 0P |
8± 1P |
49 ± 8P,M |
1 ± 1P |
1± 2P |
+ |
5000 |
0 ± 0P |
1± 1P |
44 ± 5P,M |
0 ± 0P |
0± 0P |
Positive controls, +S9 |
Name |
2-AA |
2-AA |
2-AA |
2-AA |
2-AA |
Concentrations (μg/plate) |
2.5 |
2.5 |
10 |
2.5 |
2.5 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
335 ± 38 |
3122± 296 |
321 ± 34 |
155 ± 26 |
3640± 482 |
|
NaN3: Sodium azide
4-NOPD: 4-nitro-o-phenylene-diamine
MMS: Methylmethanesulfonate
2-AA: 2-aminoanthracene
M: Manual count
P: Precipitate
Table 2. Test results of main test 2 (preincubation).
With or without S9-Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation) |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA1535 |
TA100 |
WP2 uvrA |
TA1537 |
TA98 |
||
- |
0 (DMSO) |
13 ± 4 |
128± 15 |
37 ± 14 |
10 ± 3 |
33± 6 |
- |
0 |
13 ± 3 |
173± 10 |
34 ± 8 |
13 ± 1 |
31± 5 |
- |
1 |
10 ± 4 |
111± 2 |
- |
10 ± 3 |
25± 6 |
- |
3 |
11 ± 4 |
142± 27 |
- |
6 ± 1 |
30± 2 |
- |
10 |
13 ± 5 |
134± 27 |
- |
9 ± 3 |
32± 3 |
- |
33 |
11 ± 1 |
139± 10 |
41 ± 2 |
8 ± 3 |
37± 9 |
- |
100 |
10 ± 4 |
122± 18 |
34 ± 1 |
5 ± 3 |
23± 1 |
- |
333 |
13 ± 2 |
109± 9 |
42 ± 6 |
6 ± 1 |
32± 10 |
- |
1000 |
3 ± 1 |
26± 15 |
36 ± 5 |
6 ± 3 |
12± 3 |
- |
2500 |
4 ± 3 |
18± 4R |
36 ± 5 |
6 ± 1 |
2± 3 |
- |
5000 |
- |
- |
24 ± 7M,R |
- |
- |
Positive controls, –S9 |
Name |
NaN3 |
NaN3 |
MMS |
4-NOPD |
4-NOPD |
Concentrations (μg/plate) |
10 |
10 |
2.0 µL |
50 |
10 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
1200 ± 44 |
2482± 103 |
547 ± 18 |
73 ± 17 |
419± 19 |
|
+ |
0 (DMSO) |
14 ± 4 |
113± 8 |
56 ± 7 |
9 ± 1 |
42± 2 |
+ |
0 |
13 ± 4 |
159± 14 |
58 ± 6 |
8 ± 1 |
43± 9 |
+ |
3 |
9 ± 2 |
100± 2 |
- |
12 ± 3 |
35± 6 |
+ |
10 |
14 ± 1 |
102± 9 |
- |
10 ± 5 |
43± 7 |
+ |
33 |
11 ± 5 |
105± 6 |
48 ± 7 |
7 ± 1 |
41± 6 |
+ |
100 |
12 ± 4 |
92± 5 |
47 ± 3 |
11 ± 3 |
30± 3 |
+ |
333 |
10 ± 2 |
87± 8 |
58 ± 12 |
10 ± 2 |
37± 4 |
+ |
1000 |
8 ± 1 |
66± 3 |
65 ± 10 |
11 ± 3 |
19± 4 |
+ |
2500 |
6 ± 1P |
42± 11P |
85 ± 14P |
2 ± 2P |
5± 4P |
+ |
5000 |
6 ± 3P |
0± 0P |
86 ± 21P |
0 ± 0P |
0± 0P |
Positive controls, +S9 |
Name |
2-AA |
2-AA |
2-AA |
2-AA |
2-AA |
Concentrations (μg/plate) |
2.5 |
2.5 |
10 |
2.5 |
2.5 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
334 ± 23 |
3303± 232 |
333 ± 2 |
148 ± 17 |
4081± 283 |
|
NaN3: Sodium azide
2-AA: 2-aminoanthracene
4-NOPD: 4-nitro-o-phenylene-diamine
MMS: Methylmethanesulfonate
M: Manual count
P: Precipitate
R: Reduced background growth
Table 3. Reduction in the number of spontaneous revertants (below the induction factor of 0.5) in Experiment I and II at different concentrations (µg/plate).
Strain |
Experiment I |
Experiment II |
||
|
without S9 mix |
with S9 mix |
without S9 mix |
with S9 mix |
TA 1535 |
1000 - 5000/ |
1000 - 5000 |
1000 - 2500 |
2500 - 5000 |
TA 1537 |
1000 - 5000/ |
2500 - 5000 |
/ |
2500 - 5000 |
TA 98 |
1000 - 5000/ |
2500 - 5000 |
1000 - 2500 |
2500 - 5000 |
TA 100 |
1000 - 5000 |
2500 - 5000 |
1000 - 2500 |
2500 - 5000 |
WP2 uvrA |
/ |
/ |
/ |
/ |
/ = no toxic effects
Table 1: Experiment I - 4 h exposure - Without Metabolic Activation
Concentration |
Rel. cloning efficiency I |
Rel. cell density |
Rel. cloning efficiency II |
Mutant colonies per 106cells |
Induction factor |
Culture I |
|||||
0 (DMSO) |
100.0 |
100.0 |
100.0 |
16.7 |
1.0 |
30 |
61.4 |
Culture was not continued# |
|||
60 |
62.0 |
121.0 |
103.7 |
15.2 |
0.9 |
120 |
70.6 |
127.4 |
102.4 |
4.3 |
0.3 |
240 |
66.3 |
126.8 |
90.9 |
12.6 |
0.8 |
480 (PS) |
3.3 |
93.7 |
101.9 |
8.2 |
0.5 |
720 (PS) |
37.8 |
80.5 |
91.1 |
12.1 |
0.7 |
960 (PS) |
0.4 |
Culture was not continued## |
|||
EMS, 150 |
84.0 |
94.2 |
91.6 |
148.0 |
8.9 |
Culture II |
|||||
0 (DMSO) |
100.0 |
100.0 |
100.0 |
20.5 |
1.0 |
30 |
85.5 |
Culture was not continued# |
|||
60 |
93.9 |
66.5 |
82.3 |
17.3 |
0.8 |
120 |
88.5 |
81.3 |
82.9 |
17.1 |
0.8 |
240 |
99.1 |
58.2 |
83.1 |
9.9 |
0.5 |
480 |
3.8 |
41.5 |
88.3 |
13.3 |
0.6 |
720 |
77.9 |
49.5 |
92.9 |
17.8 |
0.9 |
960 |
23.4 |
Culture was not continued## |
|||
EMS, 150 |
92.3 |
72.5 |
85.6 |
205.5 |
10.0 |
DMSO: Dimethyl sulfoxide
EMS: Ethylmethane sulfonate
(PS): Phase separation
#: Culture was not continued since a minimum of only four analysable concentrations is required.
##: Culture was not continued due to exceedingly severe cytotoxic effects.
Table 2: Experiment I - 4 h exposure - With Metabolic Activation
Concentration |
Rel. cloning efficiency I |
Rel. cell density |
Rel. cloning efficiency II |
Mutant colonies per 106cells |
Induction factor |
Culture I |
|||||
0 (DMSO) |
100.0 |
100.0 |
100.0 |
12.9 |
1.0 |
15 |
95.7 |
Culture was not continued# |
|||
30 |
105.4 |
78.3 |
99.8 |
7.8 |
0.6 |
60 |
104.8 |
74.8 |
102.7 |
7.3 |
0.6 |
120 |
111.5 |
88.0 |
87.1 |
27.3 |
2.1 |
240 |
97.7 |
105.6 |
83.1 |
11.2 |
0.9 |
360 (PS) |
17.7 |
79.7 |
96.6 |
11.3 |
0.9 |
480 (PS) |
0.0 |
Culture was not continued## |
|||
DMBA, 2.2 |
94.6 |
78.0 |
89.2 |
216.5 |
16.8 |
Culture II |
|||||
0 (DMSO) |
100.0 |
100.0 |
100.0 |
29.9 |
1.0 |
15 |
109.6 |
Culture was not continued# |
|||
30 |
101.5 |
80.4 |
90.4 |
6.1 |
0.2 |
60 |
94.6 |
131.4 |
85.9 |
6.6 |
0.2 |
120 |
84.7 |
99.2 |
94.9 |
25.1 |
0.8 |
240 |
90.8 |
101.7 |
102.4 |
14.1 |
0.5 |
360 (PS) |
9.5 |
95.5 |
92.8 |
45.8 |
1.5 |
480 (PS) |
0.0 |
Culture was not continued## |
|||
DMBA, 2.2 |
106.3 |
66.5 |
72.1 |
265.3 |
8.9 |
DMSO: Dimethyl sulfoxide
DMBA: 7,12-dimethylbenzanthracene
(PS): Phase separation
#: Culture was not continued since a minimum of only four analysable concentrations is required.
##: Culture was not continued due to exceedingly severe cytotoxic effects.
Table 3: Experiment II - 24 h exposure - Without Metabolic Activation
Concentration |
Rel. cloning efficiency I |
Rel. cell density |
Rel. cloning efficiency II |
Mutant colonies per 106cells |
Induction factor |
Culture I |
|||||
0 (DMSO) |
100.0 |
100.0 |
100.0 |
11.8 |
1.0 |
30 |
79.6 |
99.7 |
100.8 |
16.4 |
1.4 |
60 |
85.2 |
105.0 |
99.1 |
22.1 |
1.9 |
120 |
92.8 |
100.5 |
102.3 |
6.6 |
0.6 |
240 |
94.2 |
81.9 |
100.7 |
18.4 |
1.6 |
480 (PS) |
85.1 |
61.1 |
99.6 |
5.3 |
0.4 |
720 (PS) |
0.0 |
5.0 |
Culture was not continued## |
||
960 (PS) |
0.0 |
Culture was not continued## |
|||
EMS, 150 |
93.6 |
82.4 |
101.0 |
408.9 |
34.8 |
Culture II |
|||||
0 (DMSO) |
100.0 |
100.0 |
100.0 |
6.0 |
1.0 |
30 |
91.8 |
97.3 |
101.9 |
6.9 |
1.1 |
60 |
89.5 |
93.3 |
101.3 |
17.7 |
2.9 |
120 |
90.6 |
81.9 |
101.3 |
16.5 |
2.7 |
240 |
83.7 |
72.3 |
96.2 |
8.3 |
1.4 |
480 (PS) |
61.3 |
68.3 |
96.8 |
11.3 |
1.9 |
720 (PS) |
0.0 |
5.3 |
Culture was not continued## |
||
960 (PS) |
0.0 |
Culture was not continued## |
|||
EMS, 150 |
89.7 |
75.3 |
84.8 |
362.1 |
60.0 |
DMSO: Dimethyl sulfoxide
EMS: Ethylmethane sulfonate
(PS): Phase separation
##: Culture was not continued due to exceedingly severe cytotoxic effects.
Table 4: Experiment II - 4 h exposure - With Metabolic Activation
Concentration |
Rel. cloning efficiency I |
Rel. cell density |
Rel. cloning efficiency II |
Mutant colonies per 106cells |
Induction factor |
Culture I |
|||||
0 (DMSO) |
100.0 |
100.0 |
100.0 |
17.7 |
1.0 |
15 |
97.6 |
117.8 |
98.0 |
23.4 |
1.3 |
30 |
94.6 |
95.8 |
96.3 |
17.2 |
1.0 |
60 |
85.2 |
122.1 |
95.4 |
14.5 |
0.8 |
120 |
85.8 |
60.0 |
97.1 |
31.6 |
1.8 |
180 |
0.0 |
48.8 |
96.8 |
41.0 |
2.3 |
240 (PS) |
0.0 |
Culture was not continued## |
|||
300 (PS) |
0.0 |
Culture was not continued## |
|||
360 (PS) |
0.0 |
Culture was not continued## |
|||
DMBA, 12 |
86.6 |
97.8 |
94.7 |
137.7 |
7.8 |
Culture II |
|||||
0 (DMSO) |
100.0 |
100.0 |
100.0 |
10.8 |
1.0 |
15 |
69.8 |
100.3 |
97.4 |
20.8 |
1.9 |
30 |
66.1 |
113.7 |
93.5 |
23.4 |
2.2 |
60 |
64.7 |
105.2 |
91.3 |
12.1 |
1.1 |
120 |
46.8 |
98.4 |
113.5 |
14.1 |
1.3 |
180 |
0.0 |
68.5 |
93.3 |
28.0 |
2.6 |
240 (PS) |
0.0 |
5.1 |
Culture was not continued## |
||
300 (PS) |
0.0 |
Culture was not continued## |
|||
360 (PS) |
0.0 |
Culture was not continued## |
|||
DMBA, 12 |
67.6 |
69.7 |
95.5 |
180.0 |
16.7 |
DMSO: Dimethyl sulfoxide
DMBA: 7,12-dimethylbenzanthracene
(PS): Phase separation
#: Culture was not continued since a minimum of only four analysable concentrations is required.
No relevant and reproducible increase in mutant colony numbers/106cells was observed in the main experiments up to the maximum concentration. The threshold of three times the mutation frequency of the corresponding solvent control was not reached or exceeded.
In the second culture of Experiment I the mutant colonies/106cells exceeded the range of the historical solvent control data (2.4 - 44.2 mutant colonies/106cells) in the presence of metabolic activation at 360 µg/mL (45.8 mutant colonies/106cells). However, the threshold of three times the mutation frequency of the corresponding solvent control was neither reached nor exceeded and there was no dose dependent increase as indicated by the lacking statistical significance.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in culture I of the second experiment with metabolic activation. This isolated trend was judged as biologically irrelevant as the mutation frequency remained within the historical solvent control range at all evaluated concentrations.
EMS and DMBA were used as positive controls and showed a distinct increase in induced mutant colonies.
Table 1: Results of Experiment 1
Test item |
Concentration in µg/mL |
Proliferation index CBPI |
Number of cells with MN in % |
Exposure period 4 h, fixation time 40 h, without S9 mix |
|||
DMSO |
0.5% (v/v) |
1.93 |
0.50 |
MMC |
1.0 |
1.44 |
16.75S |
Test substance |
204.9 |
189 |
0.20 |
358.6 |
1.89 |
0.50 |
|
627.6PS |
1.90 |
0.40 |
|
Exposure period 4 h, fixation time 40 h, with S9 mix |
|||
DMSO |
0.5% (v/v) |
1.88 |
0.50 |
CPA |
15 |
1.35 |
9.60S |
Test substance |
204.9 |
1.88 |
0.50 |
358.6 |
1.85 |
0.50 |
|
627.6PS |
1.83 |
0.60 |
|
CPA: Cyclophosphamide
DMSO:Dimethylsulfoxide
MMC: Mitomycin C
PS: Phase separation occurred at the end of the treatment
S:The number of micronucleated cells is statistically significantly higher than corresponding control values.
Table 2: Results of Experiment 2
Test item |
Concentration in µg/mL |
Proliferation index CBPI |
Number of cells with MN in % |
Exposure period 20 h, fixation time 40 h, without S9 mix |
|||
DMSO |
0.5% (v/v) |
1.95 |
0.15 |
Demecolcin |
125.0 ng |
1.55 |
2.15S |
Test substance |
204.9 |
1.93 |
0.25 |
358.6 |
1.90 |
0.30 |
|
627.6PS |
1.93 |
0.15 |
|
DMSO:Dimethylsulfoxide
PS: Phase separation occurred at the end of the treatment
S:The number of micronucleated cells is statistically significantly higher than corresponding control values.
Either demecolcin, MMC or CPA were used as positive controls and showed distinct increases in cells with micronuclei.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Gene mutation in bacteria
A bacterial gene mutation assay with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (2015). In two independent experiments, the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and the Escherichia coli strain WP2 uvrA were exposed to the test substance using either the preincubation or the plate incorporation method. Each concentration, including the controls, was tested in triplicate. To evaluate the toxicity of the test substance a pre-experiment was performed with all strains used. Test substance concentrations of 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate were selected for the incubation with and without metabolic activation (plate incorporation test). The pre-experiment was reported as Experiment I. In Experiment II (preincubation test) the concentration range of the test substance was 1 – 5000 µg/plate. No precipitation of the test substance was observed in the overlay agar in the test tubes. Precipitation of the test substance in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate in the presence of S9 mix in both experiments. The plates incubated with the test substance showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. Only in Experiment II reduced background growth was observed in the absence of S9 mix at 2500 µg/plate in strain TA 100 and at 5000 µg/plate in strain WP2 uvrA. Toxic effects, evident as a reduction in the number of revertants, occurred in all Salmonella strains with and without metabolic activation. In the concentration range investigated, the test substance did not induce a significant increase in the mutation frequency of the tester strains in the presence and absence of a metabolic activation system. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. Under the conditions of this experiment, the test substance did not show mutagenicity in the selected S. typhimurium strains and in the E. coli strain in the presence and absence of metabolic activation.
Gene mutation in mammalian cells
The mutagenic activity of the test substance was evaluated in an in vitro mammalian cell gene mutation test according to OECD Guideline 476 and in compliance with GLP (2015). The test substance was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. The study was performed in two independent experiments, using identical experimental procedures. In Experiment I, cells were exposed for 4 h to the test substance up to concentrations of 360 and 720 µg/mL in the presence and absence of metabolic activation, respectively. Experiment II was performed with a treatment time of 4 h with and 24 h without metabolic activation up to concentrations of 180 and 480 µg/mL, respectively. Phase separation was observed after 4 h treatment at 480 µg/mL and above in Experiment I without metabolic activation and at 360 µg/mL with metabolic activation. In Experiment II, phase separation occurred at 480 µg/mL without metabolic activation. Relevant cytotoxic effects occurred in the first experiment at 480 µg/mL and above without metabolic activation and at 360 µg/mL with metabolic activation. In the second experiment cytotoxicity was noted at 180 µg/mL with metabolic activation. No relevant and reproducible increase in mutant colony numbers/106cells was observed in the main experiments up to the maximum concentration. The mutant frequency of the corresponding solvent control was not reached or exceeded. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies. In conclusion the test substance did not induce gene mutations at the HPRT locus in V79 cells under the experimental conditions reported. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.
Cytogenicity in mammalian cells
The potential of the test substance to induce mirconuclei was investigated in an in vitro mammalian cell micronucleus test in cultured peripheral human lymphocytes performed according to OECD Guideline 487 and GLP (2015). The test substance was dissolved in DMSO and two independent experiments were performed. In Experiment I, cells were incubated for 4 h with and without metabolic activation up to concentration of 1922 µg/mL. In Experiment II, cells were incubated for 20 h without metabolic activation up to 1922 µg/mL. The cells were prepared 40 h after start of treatment. In each experimental group two parallel cultures were analysed and at least 1000 binucleate cells per culture were evaluated for cytogenetic damage. In Experiment I and II phase separation of the test substance in the culture medium was observed at 627.6 µg/mL and above in the absence and presence of metabolic activation. No relevant influence on osmolarity or pH was observed. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. No increase in the number of micronucleated cells was observed after treatment with the test substance with and without metabolic activation. Mitomycin C, demecolcin and cyclophosphamide were used as positive controls and induced statistically significant increases in cells with micronuclei. In conclusion, the test substance did not induce micronuclei in the in vitro micronucleus test in human lymphocytes under the experimental conditions reported. Therefore, the test substance is considered to be non-clastogenic and non-aneugenic in this in vitro micronucleus test, when tested up to cytotoxic or the highest evaluable concentration.
Justification for classification or non-classification
The available data on genetic toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
