Registration Dossier
Registration Dossier
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EC number: 272-657-3 | CAS number: 68901-15-5
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 1535, TA 1537, TA 98 and TA 100 and TA 102 with and without metabolic activation
HPRT (OECD 476): negative in V79 cells with and without metabolic activation
Micronucleus test (OECD 487): negative in 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:
- 24 Aug - 24 Sep 1999
- 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:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 1992
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbitone/ß-naphthoflavone (80/100 mg/kg bw/d, oral)
- Test concentrations with justification for top dose:
- Preliminary toxicity study:
0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation for TA100
First experiment:
15, 50, 150, 500, 1500 and 5000 µg/plate without metabolic activation for TA98
50, 150, 500, 1500 and 5000 µg/plate without metabolic activation for TA100, TA1535, TA102 and TA1537
5, 15, 50, 150, 500, 1500 and 5000 µg/plate with metabolic activation for TA1535
5, 15, 50, 150, 500 and 1500 µg/plate with metabolic activation for TA100, TA102, TA98 and TA1537
Second experiment:
15, 50, 150, 500, 1500 and 5000 µg/plate without metabolic activation for all strains
5, 15, 50, 150, 500 and 1500 µg/plate with metabolic activation for all strains - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- benzo(a)pyrene
- mitomycin C
- other: 2-Aminoanthracene (2AA); 1,8- dihydroxyanthraquinon (DANTHRON)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation) in experiment 1 and 2
DURATION
- Exposure duration: approx. 48 h
NUMBER OF REPLICATIONS: triplicates each in 2 independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: reduction in the bacterial background lawn and/or a reduction in the frequency of revertant colonies - Evaluation criteria:
- The test material may be considered to be positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least one strain of bacteria. - Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- starting at 500 µg/plate with and without S9-mix
- Vehicle controls validity:
- valid
- 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:
- cytotoxicity
- Remarks:
- starting at 500 µg/plate with and without S9-mix
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- starting at 500 µg/plate with and without S9-mix
- Vehicle controls validity:
- valid
- 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:
- cytotoxicity
- Remarks:
- starting at 500 µg/plate with and without S9-mix
- Vehicle controls validity:
- valid
- 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:
- cytotoxicity
- Remarks:
- starting at 500 µg/plate with and without S9-mix
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
RANGE-FINDING/SCREENING STUDIES:
In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material. The test material was toxic at 5000 µg/plate to the strain of Salmonella used (TA100). The test material formulation and the S9-mix used in this experiment were both shown to be sterile.
ADDITIONAL INFORMATION ON CYTOTOXICITY: The test material caused a visible reduction in the bacterial background lawn and/or a reduction in the frequency of revertant colonies in all of the tester strains both with and without metabolic activation. The first evidence of a weakened bacterial background lawn was observed at 500 µg/plate. Generally, the test material induced a greater toxic response after the addition of S9-mix. The sensitivity of the bacterial strains to the toxicity of the test material varied between strains and between exposures with or without S9-mix. The test material was, therefore, tested up to its toxic limit. - Conclusions:
- Under the conditions of the Ames Assay the substance was not mutagenic in any of the five strains (TA1535, TA1537, TA98, TA100 and TA102) with and without metabolic activation tested.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 06 Aug - 21 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, Energie, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
- Type of assay:
- 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: 15.5, 31, 62, 123.9, 247.9, 495.8, 991.5 and 1983 µg/mL without metabolic activation
4 h treatment: 15.5, 31, 62, 123.9, 247.9, 495.8, 991.5 and 1983 µg/mL with metabolic activation
Experiment 1:
4 h treatment: 15.5, 31, 62, 124, 248, 372 and 496 µg/mL without metabolic activation
4 h treatment: 7.8, 15.5, 31, 62, 124, 186 and 248 µg/mL with metabolic activation
Experiment 2:
24 h treatment: 31, 62, 124, 248, 496 and 744 µg/mL without metabolic activation
4 h treatment: 15.5, 31, 62, 124, 155 and 186 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 to 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 186 µg/mL in Exp. I and 124 µg/mL and above in Exp. II following 4 h treatment with S9; at 372 µg/mL in Exp. I and at 496 µg/mL and above in Exp. II following 4 and 24 h treatment, respectively, without S9
- 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 991.5 µg/mL and above after 4 h treatment with and without metabolic activation. Following 24 h treatment phase separation was observed at 495.8 µg/mL and above.
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 15.5 µg/mL and 1983 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 (99.97%) and the molecular weight (198.29 g/mol) of the test item. Strong cytotoxic effects occurred after 4 h treatment at 247.9 µg/mL and above with and without metabolic activation. Following 24 h treatment, severe cytotoxicity was noted at 991.5 µg/mL and above.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
The cultures at the highest concentration with metabolic activation were not continued based on exceedingly severe cytotoxic effects in Experiment I. In Experiment II the cultures at the highest concentration without metabolic activation were not continued for the same reason. - 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:
- 22 July - 08 Sep 2015
- 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)
- Version / remarks:
- adopted 26 September 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: human
- 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 phytohemagglutinin (PHA) to the culture medium for a period of 48 h.
- 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 1:
4 h treatment: 12.9, 22.5, 39.5, 69.0, 120.8, 211.4*, 370*, 647.5*, 1133.1 and 1983 µg/mL with and without metabolic activation
Since the cultures fulfilled the requirements for cytogenetic evaluation, the preliminary test was designated Experiment 1.
Experiment 2:
20 h treatment: 12.9, 22.5, 39.5, 69.0, 120.8, 211.4*, 370*, 647.5*, 1133.1 and 1983 µg/mL without metabolic activation
* evaluated for cytogenetic damage - Vehicle / solvent:
- - Vehicle/solvent used: DMSO (0.5% (v/v)
- Justification for choice of solvent/vehicle: The solvent was chosen to 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
- Preparation time (start of exposure up to fixation or harvest preparation 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: two parallel cultures in 2 independent experiments
NUMBER OF CELLS EVALUATED: 1000 binucleated cells per culture
DETERMINATION OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index (CBPI) - Evaluation criteria:
- A test substance is considered to be negative if:
− none of the test substance concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− there is no concentration-related increase
− the results in all evaluated test substance concentrations should be within the range of the laboratory historical solvent control data
A test substance is considered to be positive if:
− at least one of the test substance 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:
- Statistical significance was confirmed by means of the Chi square test.
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- 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.
- Other confounding effects: Phase separation of the test substance was observed at the end of treatment at 647.5 µg/mL and above with and without metabolic activation.
RANGE-FINDING/SCREENING STUDIES:
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterized by the percentages of reduction in the CBPI in comparison with the controls (% cytostasis) by counting 500 cells per culture. Since the cultures fulfilled the requirements for cytogenetic evaluation, the preliminary test was designated Experiment 1.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
No cytotoxicity was observed up to the highest evaluated 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 experiment 1
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 |
|||||
TA100 |
TA1535 |
TA102 |
TA98 |
TA1537 |
||
– |
0 |
128 |
20 |
247 |
24 |
7 |
– |
0 (DMSO) |
114 ± 13.7 |
30 ± 3.5 |
317 ± 36.4 |
28 ± 3.5 |
13 ± 2.3 |
– |
15 |
NT |
NT |
NT |
25 ± 4.0 |
NT |
– |
50 |
109 ± 4.4 |
19 ± 5.5 |
278 ± 3.6 |
22 ± 3.2 |
11 ± 2.9 |
– |
150 |
112 ± 12.0 |
20 ± 2.6 |
281 ± 31.8 |
31 ± 6.5 |
12 ± 4.5 |
– |
500 |
105 ± 2.1 |
15 ± 5.5 |
279 ± 22.2 |
28 ± 2.0 |
11 ± 1.0 |
– |
1500 |
67 ± 6.1 |
5 ± 1.7 |
107 ± 12.1 |
10 ± 2.9 |
5 ± 4.7 |
– |
5000 |
0 |
0 |
0T |
0 |
0 |
Positive controls, –S9 |
Name |
ENNG |
ENNG |
MMC |
4-NQO |
9-AA |
Concentrations (μg/plate) |
3 |
5 |
0.5 |
0.2 |
80 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
539 ± 64.2 |
222 ± 39.2 |
751 ± 184.5 |
171 ± 19.0 |
784 ± 250.9 |
|
+ |
0 |
143 |
22 |
310 |
- |
10 |
+ |
0 (DMSO) |
145 ± 3.0 |
12 ± 3.5 |
310 ± 28.7 |
39 ± 1.7 |
16 ± 2.6 |
+ |
5 |
119 ± 9.8 |
12 ± 0.6 |
284 ± 63.4 |
40 ± 3.1 |
17 ± 7.8 |
+ |
15 |
118 ± 6.4 |
17 ± 4.5 |
318 ± 51.8 |
27 ± 8.1 |
15 ± 6.7 |
+ |
50 |
120 ± 14.7 |
10 ± 1.7 |
333 ± 21.7 |
29 ± 2.5 |
9 ± 2.1 |
+ |
150 |
98 ± 4.7 |
13 ± 2.6 |
296 ± 5.8 |
31 ± 4.0 |
10 ± 2.0 |
+ |
500 |
85 ± 6.1 |
6 ± 2.6 |
0V |
12 ± 3.8 |
0V |
+ |
1500 |
0V |
0V |
0V |
0V |
0T |
+ |
5000 |
NT |
0T |
NT |
NT |
NT |
Positive controls, +S9 |
Name |
2-AA |
2-AA |
DAN |
B[a]P |
2-AA |
Concentrations (μg/plate) |
1 |
2 |
10 |
5 |
2 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
1770 ± 182.4 |
192 ± 16.2 |
707 ± 65.6 |
255 ± 6.7 |
685 ± 40.8 |
|
DMSO: Dimethylsulphoxide
NT: Not tested at this dose level
2-AA: 2-aminoanthracene
9-AA: 9-aminoacridine
ENNG: N-ethyl-N-nitro-N-nitrosoguanidine
4-NQO: 4-nitroquinoline-N-oxide
MMC: Mitomycin C
B[a]P: Benzo(a)pyrene
DAN: 1,8-dihydroxyanthraquinone
T: Toxic
V: Very weak lawn
Table 2. Test results of experiment 2.
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 |
|||||
TA100 |
TA1535 |
TA102 |
TA98 |
TA1537 |
||
– |
0 |
145 |
20 |
306 |
32 |
12 |
– |
0 (DMSO) |
147 ± 5.7 |
20 ± 1.5 |
339 ± 3.2 |
33 ± 4.0 |
12 ± 5.9 |
– |
15 |
127 ± 17.7 |
18 ± 0.6 |
295 ± 23.0 |
38 ± 6.5 |
12 ± 4.9 |
– |
50 |
146 ± 7.6 |
18 ± 2.6 |
300 ± 16.1 |
28 ± 5.3 |
5 ± 1.7 |
– |
150 |
124 ± 4.5 |
15 ± 0.6 |
297 ± 29.4 |
22 ± 5.0 |
9 ± 4.6 |
– |
500 |
110 ± 26.5 |
10 ± 0.6 |
310 ± 14.9 |
22 ± 5.5 |
6 ± 3.1 |
– |
1500 |
63 ± 5.6 |
4 ± 2.1 |
158 ± 18.7 |
9 ± 3.5 |
6 ± 3.6 |
– |
5000 |
0 |
2 ± 1.5 |
0 |
1 ± 1.2 |
4 ± 3.2S |
Positive controls, –S9 |
Name |
ENNG |
ENNG |
MMC |
4-NQO |
9-AA |
Concentrations (μg/plate) |
3 |
5 |
0.5 |
0.2 |
80 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
471 ± 47.3 |
144 ± 20.9 |
1054 ± 39.6 |
139 ± 52.3 |
1032 ± 104.8 |
|
+ |
0 |
90 |
- |
- |
- |
- |
+ |
0 (DMSO) |
107 ± 9.5 |
17 ± 5.0 |
302 ± 17.1 |
32 ± 3.5 |
10 ± 2.3 |
+ |
5 |
103 ± 3.8 |
15 ± 0.6 |
302 ± 34.8 |
28 ± 2.6 |
10 ± 4.4 |
+ |
15 |
100 ± 4.6 |
11 ± 2.9 |
310 ± 9.2 |
28 ± 4.6 |
8 ± 0.6 |
+ |
50 |
88 ± 20.3 |
16 ± 5.7 |
289 ± 12.9 |
38 ± 7.1 |
8 ± 0.6 |
+ |
150 |
85 ± 7.6 |
15 ± 4.2 |
265 ± 11.1 |
29 ± 1.5 |
3 ± 1.0 |
+ |
500 |
59 ± 2.5 |
11 ± 0.6 |
0S |
10 ± 7.4S |
0V |
+ |
1500 |
21 ± 4.0V |
2 ± 2.1S |
0V |
9 ± 3.1S |
0T |
Positive controls, +S9 |
Name |
2-AA |
2-AA |
DAN |
B[a]P |
2-AA |
Concentrations (μg/plate) |
1 |
2 |
10 |
5 |
2 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
1277 ± 77.7 |
124 ± 7.4 |
727 ± 165.4 |
188 ± 13.2 |
169 ± 12.0 |
|
DMSO: Dimethylsulphoxide
NT: Not tested at this dose level
2-AA: 2-aminoanthracene
9-AA: 9-aminoacridine
ENNG: N-ethyl-N-nitro-N-nitrosoguanidine
4-NQO: 4-nitroquinoline-N-oxide
MMC: Mitomycin C
B[a]P: Benzo(a)pyrene
DAN: 1,8-dihydroxyanthraquinone
S: Sparse lawn
T: Toxic
V: Very weak lawn
Table 3. Historical control data
| positive control values up to and including 1997 | ||||||||||||||
| Strain | TA100 | TA98 | TA1535 | TA1538 | TA1537 | WP2uvrA | TA102 | |||||||
| S9-Mix | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 |
| Mean | 117 | 120 | 28 | 33 | 24 | 18 | 21 | 25 | 10 | 11 | 20 | 21 | 264 | 283 |
| SD | 25 | 22 | 7 | 7 | 5 | 4 | 7 | 6 | 2 | 2 | 5 | s | 30 | 33 |
| Min | 61 | 63 | 13 | 14 | 10 | 11 | 6 | 11 | 4 | 5 | 12 | 11 | 216 | 205 |
| Max | 196 | 177 | 56 | 52 | 39 | 39 | 41 | 50 | 17. | 20 | .39 | 40 | 339 | 343 |
| Values | 1603 | 863 | 1047 | 837 | 948 | 718 | 309 | 190 | 938 | 703 | 634 | 436 | 197 | 100 |
| vehicle control values up to and including 1997 | ||||||||||||||
| Strain | TA100 | TA98 | TA1535 | TA1538 | TA1537 | WP2uvrA | TA102 | |||||||
| S9-Mix | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 |
| Mean | 613 | 949 | 203 | 420 | 466 | 291 | 484 | 454 | 812 | 266 | 834 | 734 | 785 | 705 |
| SD | 176 | 190 | 57 | 113 | 208 | 64 | 146 | 131 | 201 | 90 | 197 | 211 | 172 | 127 |
| Min | 277 | 412 | 127 | 198 | 163 | 139 | 190 | 212 | 161 | 123 | 342 | 225 | 540 | 511 |
| M ax | 1126 | 1315 | 698 | 757 | 1005 | 516 | 799 | 915 | 1149 | 718 | 1209 | 1089 | 1188 | 1090 |
| Values | 195 | 195 | 194 | 195 | 180 | 181 | 93 | 92 | 182 | 183 | 162 | 157 | 76 | 72 |
| positive control values up to and including 1998 | ||||||||||||||
| Strain | TA100 | TA98 | TA1535 | TA1538 | TA1537 | WP2uvrA | TA102 | |||||||
| S9-Mix | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 |
| Mean | 124 | 125 | 31 | 36 | 26 | 18 | 26 | 28 | 11 | 14 | 23 | 25 | 262 | 285 |
| SD | 16 | 16 | 6 | 6 | 5 | 4 | 6 | 5 | 3 | 3 | 4 | 4 | 31 | 32 |
| Min | 73 | 81 | 15 | 22 | 12 | 10 | 15 | 18 | 5 | 6 | 13 | 13 | 191 | 231 |
| Max | 173 | 170 | 45 | 54 | 38 | 37 | 36 | 36 | 20 | 23 | 34 | 41 | 304 | 363 |
| Values | 342 | 339 | 337 | 340 | 341 | 331 | 16 | 16 | 323 | 325 | 287 | 285 | 20 | 22 |
| vehicle control values up to and including 1998 | ||||||||||||||
| Strain | TA100 | TA98 | TA1535 | TA1538 | TA1537 | WP2uvrA | TA102 | |||||||
| S9-Mix | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 |
| Mean | 556 | 1209 | 143 | 472 | 375 | 260 | 403 | 556 | 1052 | 298 | 851 | 837 | 863 | 747 |
| SD | 114 | 318 | 24 | 119 | 173 | 64 | 93 | 197 | 259 | 106 | 202 | 223 | 127 | 116 |
| Min | 349 | 681 | 101 | 198 | 112 | 105 | 305 | 344 | 173 | 123 | 415 | 260 | 646 | 591 |
| Max | 1147 | 2740 | 225 | 733 | 1143 | 601 | 604 | 1073 | 1912 | 652 | 1486 | 1380 | 1096 | 1096 |
| Values | 141 | 141 | 140 | 143 | 142 | 141 | 12 | 12 | 142 | 141 | 138 | 138 | 24 | 25 |
SD = standard deviation
Min = minimal value
Max = maximal value
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 10E6 cells |
Induction factor |
Culture I |
|||||
0 (DMSO) |
100.0 |
100.0 |
100.0 |
9.6 |
1.0 |
15.5 |
97.5 |
117.8 |
Culture was not continued# |
||
31 |
98.3 |
120.4 |
93.9 |
8.9 |
0.9 |
62 |
93.0 |
112.2 |
95.2 |
17.1 |
1.8 |
124 |
83.4 |
119.2 |
86.7 |
16.1 |
1.7 |
248 |
56.9 |
98.3 |
91.2 |
18.5 |
1.9 |
372 |
2.5 |
10.6 |
61.7 |
11.9 |
1.2 |
496 |
0.0 |
Culture was not continued## |
|||
EMS, 150 |
92.6 |
89.6 |
91.5 |
153.2 |
16.0 |
Culture II |
|||||
0 (DMSO) |
100.0 |
100.0 |
100.0 |
5.0 |
1.0 |
15.5 |
101.9 |
82.1 |
Culture was not continued# |
||
31 |
99.6 |
93.8 |
86.2 |
12.5 |
2.5 |
62 |
90.9 |
132.2 |
88.6 |
14.3 |
2.9 |
124 |
85.8 |
75.5 |
89.3 |
18.9 |
3.8 |
248 |
54.0 |
69.5 |
90.3 |
20.8 |
4.2 |
372 |
4.4 |
17.9 |
67.3 |
6.1 |
1.2 |
496 |
0.0 |
Culture was not continued## |
|||
EMS, 150 |
91.9 |
58.0 |
90.9 |
182.7 |
36.9 |
DMSO: Dimethyl sulfoxide
EMS: Ethylmethane sulfonate
#: 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 10E6 cells |
Induction factor |
Culture I |
|||||
0 (DMSO) |
100.0 |
100.0 |
100.0 |
17.8 |
1.0 |
7.8 |
105.0 |
95.5 |
Culture was not continued# |
||
15.5 |
107.9 |
106.5 |
85.4 |
13.5 |
0.8 |
31 |
99.3 |
101.3 |
94.6 |
10.4 |
0.6 |
62 |
79.9 |
106.4 |
99.3 |
19.7 |
1.1 |
124 |
51.6 |
119.6 |
106.8 |
16.3 |
0.9 |
186 |
2.1 |
56.0 |
106.4 |
29.3 |
1.6 |
248 |
0.0 |
12.3 |
Culture was not continued## |
||
DMBA, 2.2 |
86.1 |
87.5 |
86.7 |
210.9 |
11.8 |
Culture II |
|||||
0 (DMSO) |
100.0 |
100.0 |
100.0 |
17.8 |
1.0 |
7.8 |
106.8 |
98.5 |
Culture was not continued# |
||
15.5 |
102.3 |
105.0 |
102.9 |
18.5 |
1.2 |
31 |
102.5 |
95.2 |
98.9 |
20.3 |
1.3 |
62 |
82.0 |
90.4 |
97.6 |
15.4 |
1.0 |
124 |
54.1 |
83.2 |
101.1 |
15.9 |
1.0 |
186 |
4.1 |
46.7 |
93.3 |
22.5 |
1.5 |
248 |
0.0 |
7.6 |
Culture was not continued## |
||
DMBA, 2.2 |
80.7 |
105.6 |
94.2 |
144.1 |
9.5 |
DMSO: Dimethyl sulfoxide
DMBA: 7,12-dimethylbenzanthracene
#: 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 10E6 cells |
Induction factor |
Culture I |
|||||
0 (DMSO) |
100.0 |
100.0 |
100.0 |
16.5 |
1.0 |
31 |
98.8 |
89.4 |
94.3 |
8.5 |
0.6 |
62 |
91.8 |
68.9 |
87.6 |
12.9 |
0.9 |
124 |
94.3 |
105.8 |
83.4 |
22.5 |
1.5 |
248 |
91.5 |
54.0 |
83.9 |
13.4 |
0.9 |
496 |
48.7 |
80.1 |
80.8 |
12.1 |
0.8 |
744 |
0.0 |
0.0 |
Culture was not continued## |
||
EMS, 150 |
93.6 |
84.9 |
72.8 |
392.7 |
26.8 |
Culture II |
|||||
0 (DMSO) |
100.0 |
100.0 |
100.0 |
14.9 |
1.0 |
31 |
98.5 |
123.0 |
105.6 |
15.3 |
0.8 |
62 |
94.9 |
81.9 |
103.1 |
13.7 |
0.7 |
124 |
95.9 |
116.0 |
100.7 |
28.1 |
1.5 |
248 |
90.3 |
118.8 |
96.3 |
13.4 |
0.7 |
496 |
58.1 |
87.1 |
99.4 |
15.3 |
0.8 |
744 |
0.0 |
3.3 |
Culture was not continued## |
||
EMS, 150 |
93.2 |
108.8 |
97.6 |
301.4 |
16.0 |
DMSO: Dimethyl sulfoxide
EMS: Ethylmethane sulfonate
##: 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 10E6 cells |
Induction factor |
Culture I |
|||||
0 (DMSO) |
100.0 |
100.0 |
100.0 |
12.6 |
1.0 |
15.5 |
97.5 |
Culture was not continued# |
|||
31 |
97.1 |
108.8 |
106.3 |
11.9 |
0.9 |
62 |
91.9 |
88.1 |
100.5 |
12.0 |
0.9 |
124 |
33.4 |
90.7 |
99.3 |
10.5 |
0.8 |
155 |
20.7 |
94.8 |
96.8 |
9.2 |
0.7 |
186 |
0.0 |
32.1 |
97.5 |
12.1 |
0.9 |
DMBA, 2.2 |
93.0 |
76.6 |
92.9 |
186.4 |
14.5 |
Culture II |
|||||
0 (DMSO) |
100.0 |
100.0 |
100.0 |
11.7 |
1.0 |
15.5 |
101.3 |
Culture was not continued# |
|||
31 |
103.9 |
95.6 |
76.7 |
32.8 |
2.2 |
62 |
92.0 |
84.9 |
63.6 |
21.7 |
1.4 |
124 |
39.3 |
85.6 |
89.7 |
27.0 |
1.8 |
155 |
25.4 |
87.8 |
102.9 |
17.9 |
1.2 |
186 |
0.0 |
41.1 |
99.8 |
15.3 |
1.0 |
DMBA, 2.2 |
95.0 |
87.4 |
96.5 |
186.5 |
12.4 |
DMSO: Dimethyl sulfoxide
DMBA: 7,12-dimethylbenzanthracene
#: Culture was not continued since a minimum of only four analysable concentrations is required.
Table 5. Histrorical control data
| Number of mutant colonies per 10E6 cells | ||
| without metabolic activation (4 h treatment time) | ||
| Positive control EMS 150 µg/mL | Solvent control (medium, acetone, water, DMSO, ethanol, THF) | |
| Range | 54.88 - 889.0 | 1.6 - 45.7 |
| Mean value | 149.2 | 14.7 |
| Standard deviation | 99 | 7.8 |
| Number of studies | 108 | 108 |
| with metabolic activation (4 h treatment time) | ||
| Positive control DMBA 1.1 and 2.2 µg/mL | Solvent control (medium, acetone, water, DMSO, ethanol, THF) | |
| Range | 77.7 - 2042.6 | 2.4 - 44.2 |
| Mean value | 495 | 13.9 |
| Standard deviation | 302.1 | 6.9 |
| Number of studies | 108 | 108 |
| without metabolic activation 2(4 h treatment time) | ||
| Positive control EMS 150 µg/mL | Solvent control (medium, acetone, water, DMSO, ethanol, THF) | |
| Range | 108.5 - 786.1 | 2.4 - 41.8 |
| Mean value | 344.1 | 13.7 |
| Standard deviation | 126.4 | 7.1 |
| Number of studies | 103 | 103 |
No relevant and reproducible increase in mutant colony numbers/10E6 cells was observed in the main experiments up to the maximum concentration. The range of the historical solvent control data was not exceeded.
In the second culture of Experiment I the mutant colonies/10E6 cells exceeded the induction factor of three times the mutation frequency of the corresponding solvent control at 124.0 and 248.0 µg/mL in the absence of metabolic activation. The effects however, were judged as biologically irrelevant as they were clearly based upon a rather low solvent control of 5.0 mutant colonies/10E6 cells. Furthermore, the threshold was not reached at any other, even higher concentration or in the parallel culture under identical conditions.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.
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 %1) |
Exposure period 4 h, preparation interval 40 h, without S9 mix |
|||
DMSO |
0.5% (v/v) |
1.89 |
0.50 |
MMC |
1.0 |
1.35 |
9.40S |
Test substance |
211.4 |
1.85 |
0.80 |
370 |
1.82 |
0.40 |
|
647.5PS |
1.84 |
0.30 |
|
Exposure period 4 h, preparation interval 40 h, with S9 mix |
|||
DMSO |
0.5% (v/v) |
1.92 |
0.95 |
CPA |
12.5 |
1.43 |
3.05S |
Test substance |
211.4 |
1.86 |
1.00 |
370 |
1.91 |
0.55 |
|
647.5PS |
1.84 |
0.85 |
|
1) The number of micronucleated cells was determined in a sample of 2000 binucleated cells.
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 %1) |
Exposure period 20 h, preparation interval 40 h, without S9 mix |
|||
DMSO |
0.5% (v/v) |
1.58 |
0.80 |
Demecolcin |
50 ng/mL |
1.35 |
2.15S |
Test substance |
211.4 |
1.75 |
0.95 |
370 |
1.72 |
0.40 |
|
647.5PS |
1.64 |
0.55 |
|
1) The number of micronucleated cells was determined in a sample of 2000 binucleated cells.
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.
Table 3. Historical control data (2009-2013)
| Without metabolic activation | ||||
| Micronucleated cells (%) | ||||
| Concentration | No. of experiments | Range | Mean | ±Standard deviation |
| Solvent control (pulse treatment) | ||||
| Aqueous solvent 1) | 41 | 0.10 - 1.30 | 0.57 | 0.22 |
| Organic solvent 2) | 39 | 1.15 - 1.40 | 0.59 | 0.3 |
| Total | 80 | 0.10 - 1.40 | 0.58 | 0.26 |
| Solvent control (continuous treatment) | ||||
| Aqueous solvent 1) | 49 | 0.30 - 1.45 | 0.3 | 0.19 |
| Organic solvent 2) | 47 | 0.05 - 1.35 | 0.44 | 0.21 |
| Total | 96 | 0.05 - 1.45 | 0.43 | 0.2 |
| Total 3)# | 10 | 0.10 - 0.80 | 0.43 | 0.21 |
| Positive control (pulse treatment) | ||||
| Mitomycin C 0.3 - 3.0 µg/mL | 47 | 2.85 - 33.35 | 11.35 | 5.18 |
| Positive control (continuous treatment) | ||||
| Demecolcin 50 - 150 ng/mL | 52 | 1.40 - 6.85 | 3.62 | 1.05 |
| Demecolcin# 50 - 175 ng/mL | 11 | 1.30 - 5.45 | 2.79 | 1.18 |
| With metabolic activation | ||||
| Solvent control (pulse treatment) | ||||
| Aqueous solvent 1) | 79 | 0.15 - 1.70 | 0.65 | 0.27 |
| Organic solvent 2) | 70 | 0.15 - 1.65 | 0.66 | 0.29 |
| Total | 149 | 0.15 - 1.70 | 0.65 | 0.28 |
| Positive control (pulse treatment) | ||||
| CPA 7.5 - 20.0 µg/mL | 81 | 2.15 - 11.05 | 5.22 | 1.89 |
1) Aqueous solvents: DMEM/Ham’s F12, deionised water (10 % v/v)
2) Organic solvents: DMSO (0.5 or 1.0 %), acetone, ethanol and THF (0.5 %)
3) Aqueous and organic solvents
# Micronucleated mononucleate cells
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 (Bowles, 1999). In two independent experiments, the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 were exposed to the test substance suspended in DMSO using the plate incorporation method. The dose range for the first experiment was determined in a preliminary toxicity assay and ranged from 5 to 5000 µg/plate depending on bacterial strain type and presence or absence of metabolic activation. The dose range of the second experiment was based on the results observed in Experiment 1 and ranged from 5 to 5000 µg/plate depending on bacterial strain type and presence or absence of metabolic activation. The test substance caused a visible reduction in the bacterial background lawn and/or a reduction in the frequency of revertant colonies in all of the tester strains both with and without metabolic activation. The first evidence of a weakened bacterial background lawn was observed at 500 µg/plate. Generally, the test substance induced a greater toxic response after the addition of S9-mix. The sensitivity of the bacterial strains to the toxicity of the test material varied between strains and between exposures with or without S9-mix. The test substance was, therefore, tested up to its toxic limit. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. The vehicle control plates gave counts of revertant colonies within the normal range. A marked increase in the frequency of revertant colonies, both with and without metabolic activation, was observed as a result of treatment with positive control chemicals.
Under the conditions of this experiment, the test substance did not show mutagenicity in the selected S. typhimurium strains 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. Based on the results of a pre-test, cells of the first experiment were exposed to the test substance for 4 h at concentrations ranging from 31 - 372 µg/mL without metabolic activation and from 15.5 - 186 µg/mL with metabolic activation. In the second experiment cells were exposed to the test substance for 4 h at concentrations ranging from 31 - 186 µg/mL and for 24 h at concentrations ranging from 31 - 496 µg/mL. Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both cultures occurred in the first experiment at 372 μg/mL without metabolic activation and at 186 μg/mL with metabolic activation. In the second experiment cytotoxicity as described above was noted at 124 μg/mL and above with metabolic activation. In the second experiment without metabolic activation a steep gradient of cytotoxicity occurred with only moderate toxic effects at 496 μg/mL. No relevant and reproducible increase in mutant colony numbers/10E6 cells was observed in the main experiments up to the maximum concentration. The range of the historical solvent control data was not exceeded. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. 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 human lymphocytes performed according to OECD Guideline 487 and GLP (Sokolowski, 2016). The test substance was dissolved in DMSO and cytotoxicity of the test substance was investigated in a preliminary test. Cells were incubated for 4 hours with and without metabolic activation up to 1983 µg/mL (approx. 10 mM). Since the cultures fulfilled the requirements for cytogenetic evaluation, the preliminary test was designated Experiment 1. In Experiment 2, cells were incubated for 20 hours without S9 mix up to 1983 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. Phase separation of the test substance was observed at the end of treatment at 647.5 µg/mL and above with and without metabolic activation. No relevant influence of the test substance on osmolarity or pH value was observed. No cytotoxicity was observed up to the highest evaluated concentration. No biologically relevant increase in the number of micronucleated cells was observed after treatment with the test substance. 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 phase separating concentrations.
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.
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