Registration Dossier
Registration Dossier
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EC number: 226-166-6 | CAS number: 5308-25-8
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
negative Ames test, in vitro MNT and HPRT
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- (2-aminoanthracene was used as sole indicator of the efficacy of the S-9 mix)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
- Version / remarks:
- Date of deletion: 21 July 1997 (Method merged with TG 471)
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his-, trp-operon
- Species / strain / cell type:
- other: S. typhimurium TA 100, TA 1535, TA 1537, TA 98 and E. coli WP2 uvrA
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor induced rat liver S-9 mix
- Test concentrations with justification for top dose:
- 20, 100, 500, 2500, 5000 µg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Water (DMSO was used as vehicle for the positive controls)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene (2.5 µg/plate for TA100, TA98, TA1537 and TA1535, 60 µg/plate for E.coli)
- Remarks:
- with metabolic activation
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: N-methyl-N-nitro-N-nitrosoguanidine (5 µg/plate for TA100, TA1535), N-ethyl-N-nitro-N-nitrosoguanidine (10 µg/plate for E.coli), 9-aminoacridine (100 µg for TA1537), 4-nitro-o-phenylendiamine (10 µg/plate for TA98)
- Remarks:
- without metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: standard plate test and preincubation test both with and without metabolic activation
DURATION
- Preincubation period: at 37°C for the duration of 20 minutes
- Exposure duration: at 37°C for 48 - 72 hours in the dark (for standard plate test and preincubation test) - Evaluation criteria:
- In general, a substance to be characterized as positive in the bacterial tests has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results - Species / strain:
- other: S. typhimurium TA 100, TA 1535, TA 1537, TA 98 and E. coli WP2 uvrA
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- (but tested up to limit dose)
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- No precipitation of the test substance was found.
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- negative Ames test
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- BASF SE, Experimental Toxicology and Ecology, Ludwigshafen, Germany
- Type of assay:
- in vitro mammalian cell micronucleus test
- Target gene:
- not applicable
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MEM (minimal essential medium with Earle's salts) containing a L-glutamine source supplemented with
− 10% (v/v) fetal calf serum (FCS);
− 1% (v/v) penicillin/streptomycin (10000 IU / 10000 μg/mL);
− 1% (v/v) amphotericine B (250 μg/mL);
During exposure to the test substance for 4 hours only, MEM medium was used without FCS supplementation.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix (1 part S9 fraction from phenobarbital and β-naphthoflavone induced rat livers was mixed with 9 parts S9 supplement (cofactors))
- Test concentrations with justification for top dose:
- 1st Experiment:
- 4 hours exposure; 24 hours harvest time; without S9 mix: 0; 37.5; 75.0; 150.0; 300.0; 600.0 and 1200.0 μg/mL (evaluated: 0; 150.0; 300.0; 600.0 μg/mL);
- 4 hours exposure, 24 hours harvest time, with S9 mix: 0; 37.5; 75.0; 150.0; 300.0; 600.0 and 1200.0 μg/mL (evaluated: 0; 300.0; 600.0 and 1200.0 μg/mL);
2nd Experiment:
- 24 hours exposure, 24 hours harvest time, without S9 mix: 0; 37.5; 75.0; 150.0; 300.0; 600.0 and 1200.0 μg/mL (evaluated: 0; 37.5; 75.0; 150.0 μg/mL);
- 4 hours exposure, 24 hours harvest time, with S9 mix: 0; 225.0; 450.0; 900.0 and 1200.0 μg/mL (evaluated: 0; 225.0; 450.0; 900.0 and 1200.0 μg/mL); - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: culture medium (MEM)
- Justification for choice of solvent/vehicle: good solubility of the test substance in water - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Remarks:
- Without metabolic activation: 500 and 600 μg/mL EMS; with metabolic activation: 2.5 μg/mL CPP;
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 6 h
- Exposure duration: 4 h (1st experiment without / with metabolic activation; 2nd experiment with metabolic activation), 24 h (2nd experiment without metabolic activation)
- Expression time (cells in growth medium): 20 h (1st experiment without / with metabolic activation; 2nd experiment with metabolic activation), no expression time, continuous treatment (2nd experiment without metabolic activation)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h
STAIN (for cytogenetic assays): 2.6% (v/v) Giemsa/Titrisol solution pH 7.2 (20 min)
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 1000 / culture (i.e. 2000 cells / test group)
DETERMINATION OF CYTOTOXICITY
- Method: relative increase in cell count (RICC), proliferation index (PI), cell morphology - Evaluation criteria:
- Acceptance criteria:
The in vitro micronucleus assay is considered valid if the following criteria are met:
- The quality of the slides allowed the identification and evaluation of a sufficient number of analyzable cells.
- The number of cells containing micronuclei in the negative control was within the range of the historical negative control data.
- The positive control substances both with and without S9 mix induced a significant increase in the number of micronucleated cells.
Assessment criteria:
A test substance is considered "positive" if the following criteria are met:
- A significant, dose-related and reproducible increase in the number of cells containing micronuclei.
- The number of micronucleated cells exceeds both the value of the concurrent negative and the range of the historical negative control data.
A test substance generally is considered "negative" if the following criteria are met:
- The number of micronucleated cells in the dose groups is not significant increased above the concurrent negative control value and is within the range of the historical negative control data. - Statistics:
- The statistical evaluation of the data was carried out using the MUVIKE program system (BASF SE). The proportion of cells containing micronuclei was calculated for each group. A comparison of each dose group with the concurrent negative/vehicle control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test is Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided.
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (mainly in the experiments without S9 mix: 1st Experiment: at 1200 µg/mL; 2nd Experiment: at 300 µg/mL onwards)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH / osmolality: The pH value of the test substance preparation was adjusted by adding small amounts of HCl (32% (w/v) ). Thus, osmolarity and pH values were not influenced by test substance treatment.
- Water solubility: Good solubility in water
- Precipitation: In culture medium no test substance precipitation occurred up to the highest applied concentration at the end of treatment in the absence and presence of S9 mix.
RANGE-FINDING/SCREENING STUDIES: The doses/concentrations for this study were selected based on the results of a preliminary range finding test of an in vitro gene mutation test in CHO cells (HPRT locus assay) for gene mutation (50M0475/11M177).
COMPARISON WITH HISTORICAL CONTROL DATA: yes
ADDITIONAL INFORMATION ON CYTOTOXICITY: In the absence and the presence of S9 mix no cytotoxicity indicated by reduced PI values was observed at the test groups scored for cytogenetic damage. In addition, in 1st experiment in the absence of S9 mix growth inhibition indicated by reduced cell counts was observed at 1200.0 μg/mL (RICC: 45.6%). Additionally, in the 2nd experiment after 24 hours continous treatment without S9 mix the cell numbers were clearly reduced from 600.0 μg/mL (RICC: 25.1%) onwards. Besides, in both experiments after metabolic activation at 4 hours exposure period no growth inhibition was observed up to the highest required concentration. In the experimental parts without S9 mix cell morphology and the quality of the cells on the slides was the limiting factor for dose selection for light microscopical evaluation of genotoxicity. Poor slide quality as indication of test substance toxicity was obtained in the 1st experiment after 4 hours treatment in the highest dose (1200 μg/mL) and in the 2nd experiment after 24 hours treatment from 300 μg/mL onwards. In the presence of a metabolic system the quality to the cells on the slide was not influenced. However, the cell morphology was slightly influenced from 600 μg/mL onwards in the 1st experiment and at 1200 μg/mL in the 2nd experiment. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- negative in vitro MNT
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- 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:
- (21.07.1997)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Experimental Toxicology and Ecology, Ludwigshafen, Germany
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- - Type and identity of media:
All media were supplemented with: 1% penicillin/streptomycin and 1% amphotericine B; Treatment medium (4-hour exposure period): Ham's F12 medium containing stable glutamine and hypoxanthine; culture medium and treatment medium (24-hour exposure): Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10% fetal calf serum (FCS); pretreatment medium ("HAT" medium): Ham's F12 medium supplemented with hypoxanthine, aminopterin, thymidine, 10% fetal calf serum; selection medium ("TG" medium): Hypoxanthine-free Ham's F12 medium supplemented with 6-thioguanine, 1% stable glutamine;
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- liver S9 mix from phenobarbital and β-naphthoflavone induced rats
- Test concentrations with justification for top dose:
- 1st Experiment:
- without / with S9 mix (4-hour exposure period): 0, 75, 150, 300, 600, 1200 μg/mL (evaluated concentrations: 0, 150, 300, 600, 1200 μg/mL)
2nd Experiment:
without S9 mix (24-hour exposure period): 0, 50, 100, 200, 400, 800, 1200 μg/mL (evaluated concentrations: 0, 200, 400, 800, 1200 μg/mL)
with S9 mix (4-hour exposure period): 0, 200, 400, 800, 1200 μg/mL (all concentrations evaluated) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: water medium (Ham's F12)
- Justification for choice of solvent/vehicle: good solubility of the test substance in water medium (Ham's F12) - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- without metabolic activation, 300 µg/mL;
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 3-methylcholanthrene
- Remarks:
- with metabolic activation, 20 µg/mL;
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 20 - 24 h
- Exposure duration: 4 h (1st experiment: with and without metabolic activation; 2nd experiment: with metabolic activation); 24 h (2nd experiment: without metabolic activation)
- Expression time (cells in growth medium): 6 - 8 days
- Selection time (if incubation with a selection agent): about 1 week
SELECTION AGENT (mutation assays): selection medium ("TG" medium): Hypoxanthine-free Ham's F12 medium supplemented with 6-thioguanine, 1% stable glutamine
NUMBER OF REPLICATIONS: duplicate cultures for each dose in 2 independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, cell morphology - Evaluation criteria:
- Acceptance criteria:
The HPRT assay is considered valid if the following criteria are met:
- The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50% (with and without S9 mix).
- The background mutant frequency in the negative/vehicle controls should fall within the historical negative control data range of 0 – 15.95 mutants per 10^6 clonable cells.
- The positive controls both with and without S9 mix must induce distinctly increased mutant frequencies (historical positive control data).
- At least 4 dose levels ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions should be tested. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.
Assessment criteria:
A finding is assessed as positive if the following criteria are met:
- Increase of the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and the historical negative control data range.
- Evidence of reproducibility of any increase in mutant frequencies.
- A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship.
Isolated increases of mutant frequencies above the historical negative control range (i.e. 15 mutants per 10^6 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
The test substance is considered non-mutagenic according to the following criteria:
- The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significant increased above the concurrent negative control and is within our historical negative control data range. - Statistics:
- An appropriate statistical trend test was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective negative/vehicle control groups. A trend is judged as statistically significant whenever the p-value (probability value) is below 0.10 and the slope is greater than 0. However, both, biological and statistical significance will be considered together.
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (cell morphology and attachment of the cells was adversely influenced at least at the highest concentration at the 1st Experiment (-/+ S9) and at the 2nd Experiment (-S9 mix))
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Due to a pH shift observed at the dose group 1200 μg/mL (stock solution) in the pretest, in the main experiments the pH of the stock solution was determined and adjusted case-by-case to a physiological value using small amounts of 32% (w/v) HCl. Then, the pH values were not influenced by test substance treatment in the main test.
- Effects of osmolality: not influenced by test substance treatment
- Precipitation: In the absence and the presence of S9 mix no precipitation in culture medium was observed up to the highest required test substance concentration.
RANGE-FINDING/SCREENING STUDIES: In the pretest for toxicity based on the purity 1200 μg/mL was used as top concentration both with and without S9 mix at 4-hour exposure time and without S9 mix at 24-hour exposure time. The pretest was performed following the method described for the main experiment. The cloning efficiency 1 (survival) was determined as toxicity indicator for dose selection and various parameters were checked for all or at least for some selected doses. In the pretest the parameters pH value and osmolarity were not influenced by the addition of the test substance preparation to the culture medium at the concentrations measured. However, a pH shift was observed at the highest required concentration prior to testing. Therefore, the pH of the stock solution was adjusted to a physiological value prior to application using small amounts of 32% (w/v) HCl. In culture medium no test substance precipitation occurred up to the highest concentration at the end of treatment in the absence and the presence of S9 mix. After 4 hours treatment in the absence and presence of S9 mix no cytotoxicity indicated by reduced relative cloning efficiency of about or below 20% relative survival was observed. After 24 hours treatment in the absence of S9 mix strongly reduced relative cloning efficiency of below 20% relative survival was observed after treatment with 1200 μg/mL. On the basis of the data and the observations from the pretest and taking into account the current guidelines, the doses for the main test were selected.
COMPARISON WITH HISTORICAL CONTROL DATA: In both experiments after 4 and 24 hours treatment with the test substance the values for the corrected mutation frequencies were clearly within the range of the historical negative control data.
ADDITIONAL INFORMATION ON CYTOTOXICITY: In both experiments in the presence and absence of S9 mix, no cytotoxicity concerning cloning efficiency was observed up to the top dose applied. In the 1st Experiment in the absence and presence, and in the 2nd Experiment in the absence of S9 mix after 24 treatment the morphology and attachment of the cells was adversely influenced at least at the highest applied concentration.
During the cell seeding of the cloning efficiency 2 in the 1st Experiment without metabolic activation a technical error occurred. At two dilution steps a volume of 200 μL instead of 100 μL was pipetted in the respective vessels. Therefore the cloning efficiency 2 values were by a factor of 4 higher than usual. As expected no cytotoxicity of the test substance was observed in this experimental part. Therefore, it has to be considered that this deviation to the common practize had no influence on the validity of the cytotoxicity data. For further calculation of the corrected mutation frequency a factor of 4 was included in this case only. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- negative HPRT test
Referenceopen allclose all
Standard plate test:
Dose (µg/plate) |
TA1535 |
TA100 |
TA1537 |
TA98 |
E. coli WP2 uvrA |
|||||
|
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
0 |
20±1 |
20±1 |
135±5 |
131±6 |
12±3 |
10±2 |
31±3 |
39±7 |
33±2 |
38±3 |
20 |
17±2 |
19±2 |
127±7 |
116±7 |
9±4 |
15±4 |
27±2 |
34±5 |
32±6 |
34±3 |
100 |
16±2 |
16±1 |
127±14 |
127±17 |
11±1 |
9±3 |
28±1 |
37±3 |
31±4 |
31±5 |
500 |
15±1 |
15±1 |
108±17 |
120±22 |
7±2 |
11±3 |
24±3 |
34±4 |
32±4 |
34±4 |
2500 |
13±2 |
15±3 |
98±17 |
128±10 |
9±3 |
9±3 |
19±6 |
30±4 |
38±10 |
34±2 |
5000 |
13±1 |
14±0 |
99±12 |
102±16 |
11±2 |
12±3 |
19±2 |
28±2 |
31±1 |
30±2 |
2-AA |
- |
159±30 |
- |
1298±60 |
- |
204±6 |
- |
1211±32 |
- |
233±9 |
MNNG |
1012±26 |
- |
1483±63 |
- |
- |
- |
- |
- |
- |
- |
AAC |
- |
- |
- |
- |
475±51 |
- |
- |
- |
- |
- |
NPD |
- |
- |
- |
- |
- |
- |
956±42 |
- |
- |
- |
ENNG |
- |
- |
- |
- |
- |
- |
- |
- |
517±14 |
- |
Mean ± SD
Preincubation test:
Dose (µg/plate) |
TA1535 |
TA100 |
TA1537 |
TA98 |
E coli. WP2 uvrA |
|||||
|
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
0 |
20±1 |
21±2 |
114±4 |
123±5 |
10±1 |
11±1 |
30±3 |
43±4 |
30±3 |
48±6 |
20 |
19±2 |
18±2 |
121±22 |
149±12 |
10±3 |
12±2 |
27±2 |
41±1 |
31±1 |
44±4 |
100 |
18±2 |
17±1 |
146±13 |
159±15 |
10±3 |
11±1 |
29±5 |
38±4 |
31±5 |
47±10 |
500 |
17±1 |
15±4 |
137±19 |
152±14 |
10±0 |
12±1 |
28±2 |
29±4 |
32±3 |
47±7 |
2500 |
18±2 |
16±1 |
150±2 |
158±7 |
10±1 |
10±2 |
29±3 |
31±2 |
27±2 |
42±3 |
5000 |
16±2 |
10±1 |
109±8 |
158±6 |
10±1 |
9±2 |
19±4 |
29±3 |
20±3 |
48±3 |
2-AA |
- |
136±11 |
- |
1137±90 |
- |
124±11 |
- |
1046±64 |
- |
183±12 |
MNNG |
1295±50 |
- |
1068±14 |
- |
- |
- |
- |
- |
- |
- |
AAC |
- |
- |
- |
- |
520±70 |
- |
- |
- |
- |
- |
NPD |
- |
- |
- |
- |
- |
- |
995±41 |
- |
- |
- |
ENNG |
- |
- |
- |
- |
- |
- |
- |
- |
1129±122 |
- |
Mean ± SD
2-AA: 2-aminoanthracene;
MNNG; N-methyl-N-nitro-N-nitrosoguanidine
ENNG; N-ethyl-N-nitro-N-nitrosoguanidine
NPD: 4-nitro-o-phenylendiamine
AAC: 9-aminoacridine chloride monohydrate
Results of the in vivo micronucleus assay:
Exp. |
Exposure period |
Test group |
S9 mix |
Prec.* |
Genotoxicity Micronucleated cells** [%] |
Cytotoxicity |
|
Proliferation index (PI) |
RICC*** [%] |
||||||
1 |
4 h |
Negative control |
- |
n.d. |
0.6 |
2.24 |
100.0 |
|
|
37.5 µg/mL |
- |
- |
n.d. |
n.d. |
82.8 |
|
|
75.0 µg/mL |
- |
- |
n.d. |
n.d. |
79.1 |
|
|
150.0 µg/mL |
- |
- |
0.7 |
2.03 |
89.3 |
|
|
300.0 µg/mL |
- |
- |
0.8 |
2.11 |
88.5 |
|
|
600.0 µg/mL |
- |
- |
0.6 |
2.08 |
85.8 |
|
|
1200.0 µg/mL |
- |
- |
n.s. |
n.s. |
45.6 |
|
|
Positive control |
- |
n.d. |
2.7s |
2.16 |
n.t. |
2 |
24 h |
Negative control |
- |
n.d. |
1.0 |
2.71 |
100.0 |
|
|
37.5 µg/mL |
- |
- |
0.4 |
2.31 |
85.4 |
|
|
75.0 µg/mL |
- |
- |
0.8 |
2.29 |
92.7 |
|
|
150.0 µg/mL |
- |
- |
0.4 |
2.61 |
83.5 |
|
|
300.0 µg/mL |
- |
- |
n.s. |
n.s. |
56.3 |
|
|
600.0 µg/mL |
- |
- |
n.s. |
n.s. |
25.1 |
|
|
1200.0 µg/mL |
- |
- |
n.s. |
n.s. |
-0.6 |
|
|
Positive control |
- |
n.d. |
6.8s |
2.10 |
n.t. |
1 |
4 h |
Negative control |
+ |
n.d. |
1.0 |
2.21 |
100.0 |
|
|
37.5 µg/mL |
+ |
- |
n.d. |
n.d. |
73.7 |
|
|
75.0 µg/mL |
+ |
- |
n.d. |
n.d. |
77.9 |
|
|
150.0 µg/mL |
+ |
- |
n.d. |
n.d. |
83.1 |
|
|
300.0 µg/mL |
+ |
- |
1.2 |
2.19 |
75.1 |
|
|
600.0 µg/mL |
+ |
- |
0.7 |
2.21 |
82.7 |
|
|
1200.0 µg/mL |
+ |
- |
1.3 |
2.23 |
80.1 |
|
|
Positive control |
+ |
n.d. |
2.6s |
1.93 |
n.t. |
2 |
4 h |
Negative control |
+ |
n.d. |
1.3 |
2.29 |
100.0 |
|
|
225.0 µg/mL |
+ |
- |
n.d. |
n.d. |
93.8 |
|
|
450.0 µg/mL |
+ |
- |
1.0 |
2.29 |
95.6 |
|
|
900.0 µg/mL |
+ |
- |
0.6 |
2.22 |
97.3 |
|
|
1200.0 µg/mL |
+ |
- |
0.7 |
2.25 |
85.9 |
|
|
Positive control |
+ |
- |
4.3s |
1.97 |
n.t. |
*Precipitation in culture medium at the end of exposure period
**Relative number of micronucleated cells per 2000 cells scored per test group
*** Relative increase in cell count (RICC)
s Frequency statistically significant higher than corresponding control values
n.d. Not determined
n.t. Not tested
n.s. Not scorable due to strong cytotoxicity
Table 1: Experiment I - 4 h exposure - With Metabolic Activation
Concentration |
Cloning efficiency 1 [%] |
Cloning efficiency 2 [%] |
Mutants per 1E+06 surviving cells |
Mutation factor |
0 (medium) |
100 |
100 |
2,74 |
1 |
75 |
104.4 |
n.c. |
n.c. |
n.c. |
150 |
109.4 |
106.9 |
1.38 |
0.50 |
300 |
103.5 |
112.9 |
2.35 |
0.86 |
600 |
99.7 |
108.4 |
1.10 |
0.40 |
1,200 |
98.3 |
103.4 |
1.17 |
0.43 |
MCA |
89.3 |
101.5 |
64.23 |
23.44 |
Table 2: Experiment I - 4 h exposure - Without Metabolic Activation
Concentration |
Cloning efficiency 1 [%] |
Cloning efficiency 2 [%] |
Mutants per 1E+06 surviving cells |
Mutation factor |
0 (medium) |
100 |
100 |
4.16 |
1 |
75 |
115.7 |
n.c. |
n.c. |
n.c. |
150 |
116.0 |
96.2 |
1.73 |
0.42 |
300 |
109.4 |
103.8 |
2.77 |
0.67 |
600 |
109.8 |
101.0 |
1.89 |
0.45 |
1,200 |
99.1 |
100.1 |
2.21 |
0.53 |
EMS |
116.0 |
95.3 |
99.25 |
23.86 |
Table 3: Experiment II - 4 h Exposure - With Metabolic Activation
Concentration |
Cloning efficiency 1 [%] |
Cloning efficiency 2 [%] |
Mutants per 1E+06 surviving cells |
Mutation factor |
0 (medium) |
100 |
100 |
2.08 |
1 |
200 |
102.3 |
100.8 |
2.03 |
0.98 |
400 |
94.7 |
101.4 |
1.16 |
0.56 |
800 |
96.5 |
103.6 |
3.31 |
1.59 |
1,200 |
86.6 |
106.0 |
2.73 |
1.31 |
MCA |
93.5 |
109.8 |
58.68 |
28.21 |
Table 4: Experiment II - 24 h exposure - Without Metabolic Activation
Concentration |
Cloning efficiency 1 [%] |
Cloning efficiency 2 [%] |
Mutants per 1E+06 surviving cells |
Mutation factor |
0 (medium) |
100 |
100 |
2.86 |
1 |
50 |
110.6 |
n.c. |
n.c. |
n.c. |
100 |
96.1 |
n.c. |
n.c. |
n.c. |
200 |
85.9 |
90.7 |
1.82 |
0.64 |
400 |
77.6 |
89.5 |
1.89 |
0.66 |
800 |
67.0 |
90.3 |
0.63 |
0.22 |
1,200 |
33.7 |
102.0 |
2.73 |
0.95 |
EMS |
70.6 |
56.3 |
346.21 |
121.05 |
EMS: ethyl methanesulfonate (300 µg/mL), MCA: methylcholanthrene (20 µg/mL)
n.c. Culture was not continued since a minimum of only four analyzable concentrations are required.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
no data available
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
In vitro studies
Gene mutation in bacterial cells
In a bacterial reverse mutation assay, conducted similar to OECD Guideline 471 (Bacterial Reverse Mutation Assay), the test substance was evaluated for its mutagenic potential based on the ability to induce point mutations in selected loci of Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E.coli WP2 uvrA (1998, reliability score 1). Doses of 20, 100, 500, 2500 and 5000 µg/plate were evaluated in a standard plate and in a preincubation test. Water was used as vehicle control and appropriate positive control substances were also evaluated. No effects of cytotoxicity or precipitation were observed. No dose-related biologically relevant increase or doubling in the number of his-/trp-revertants was observed in either test approaches in any of the tested strains with or without metabolic activation. According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium/E.coli reverse mutation assay under the experimental conditions chosen. The positive controls yielded the expected results.
Gene mutation in mammalian cells
The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro (2012, reliability score 1). The GLP study was conducted according to OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test; HPRT locus). Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital and β-naphthoflavone induced rats. The following doses were evaluated in this study: 1st Experiment: 0, 150, 300, 600, 1200 μg/mL (without / with S9 mix, 4-hour exposure period); 2nd Experiment: 0, 200, 400, 800, 1200 μg/mL (without S9 mix, 24-hour exposure period; with S9 mix, 4-hour exposure period). After an attachment period of 20 - 24 hours and the respective treatment period an expression phase of about 6 - 8 days and a selection period of about 1 week followed. The colonies of each test group were fixed, stained with Giemsa and counted. In the 1st Experiment and in the 2nd Experiment in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest required concentration evaluated for gene mutations. But the morphology and attachment of the cells was adversely influenced at least at the highest applied concentration in the 1st Experiment in the absence and presence, and in the 2nd Experiment in the absence of S9 mix after 24. The test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after adding a metabolizing system.
Chromosome aberration test in mammalian cells
An in vitro micronucleus test was conducted according to OECD Guideline 487 and GLP (2012, reliability score 1). The test substance was assessed in V79 cells in vitro (clastogenic or aneugenic activity) both in the absence and the presence of a metabolizing system. The following concentrations were tested and evaluated: 1st Experiment: 0, 150, 300, 600 μg/mL (without S9 mix; 4 hours exposure; 24 hours harvest time); 0, 300, 600, 1200 μg/mL (with S9 mix; 4 hours exposure; 24 hours harvest time); 2nd Experiment: 0, 37.5, 75.0, 150.0 μg/mL (without S9 mix; 24 hours exposure, 24 hours harvest time); 0, 225, 450, 900 and 1200 μg/mL (with S9 mix; 4 hours exposure, 24 hours harvest time). A sample of at least 1000 cells for each culture were analyzed for micronuclei, i.e. 2000 cells for each test group. Cytotoxicity indicated by clearly reduced relative increase in cell count or proliferation index was observed at least at the highest applied test substance concentration in the absence of S9 mix only. The test substance did not cause any relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other.
In vivo studies
No studies were conducted, because no positive results were
recorded in the in vitro studies.
Justification for classification or non-classification
The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the data, the test substance has not to be classified.
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