Registration Dossier
Registration Dossier
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EC number: 221-361-2 | CAS number: 3077-27-8
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Bacterial mutagenicity (OECD 471), Ames: negative with and without metabolic activation
Mammalian mutageniticty (OECD 490), in vitro mammalian cell gene mutation using the thymidine kinase gene: negative with and without metabolic activation
Mammalian cytogenicity (OECD 473), chromosome aberration: negative 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:
- 25 July - 11 Aug 2017
- 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:
- 21 July 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 30 May 2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, 55116 Mainz, Germany
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon
- Species / strain / cell type:
- S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102 and TA 1535
- Metabolic activation:
- with and without
- Metabolic activation system:
- Cofactor supplemented post-mitochondrial fraction (S9-mix), prepared from livers treated with Aroclor 1254 (500 mg/kg bw)
- Test concentrations with justification for top dose:
- Experiment 1a, plate incorporation method
S. typhimurium TA 97a, TA 98, TA 100, TA 102 and TA 1535: 5000, 1500, 500, 150 and 50 μg/plate with and without metabolic activation
Experiment 1b, plate incorporation method
S. typhimurium TA102: 5000 μg/plate, 1500, 500, 150, 50, 15 and 5 μg/plate with and without metabolic activation
Experiment 2, pre-incubation method
S. typhimurium TA 97a, TA 98, TA 100 and TA 1535): 5000, 2500, 1250, 625, 313, 156 and 78 μg/plate with and without metabolic activation
S. typhimurium TA 102: 500, 250, 125, 62.5, 31.3, 15.6 and 7.8 μg/plate with and without metabolic activation - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The test item was not sufficiently soluble in demineralized water, dimethyl sulfoxide (DMSO), ethanol and tetrahydrofurane. Based on these results, ethanol was used as solvent in all experiments, because the test item showed in ethanol the best result for handling in the experiments. The test suspension was homogeneous and pipettable. In addition, ethanol does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- benzo(a)pyrene
- other: 2-Amino-anthracene (2-AA): +S9: 1 µg/plate (in DMSO) for TA 97a, TA 100, TA 102, TA 1535; 4-Nitro-1,2-phenylenediamine (NPD): -S9: 20 µg/plate (in DMSO) for TA 97a, TA 98, TA 102
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation, experiments 1a and 1b) and pre-incubation (experiment 2)
DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: triplicates each in two independent experiments (experiments 1a/1b and 2)
DETERMINATION OF CYTOTOXICITY
- Method: Inspection of the bacterial background lawn and of the number of revertants - Evaluation criteria:
- The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.
A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity. - Statistics:
- The colonies were counted visually and the numbers were recorded. A validated spreadsheet was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
- Species / strain:
- S. typhimurium, other: TA 97a, TA 98, TA 100 and TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- -/+S9: 5000 and 1500 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed in any experiment at any concentration
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%) : data is summarised in "Any other information on results incl. tables" - Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 7 Aug - 9 Oct 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Version / remarks:
- adopted 29 July 2016
- Deviations:
- yes
- Remarks:
- Some of the total suspension growth (TSG) values of the solvent controls were slightly below the range specified in the guideline. In experiment II only 3 test item concentrations were evaluated.
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- adopted 30 May 2008
- Deviations:
- yes
- Remarks:
- Some of the total suspension growth (TSG) values of the solvent controls were slightly below the range specified in the guideline. In experiment II only 3 test item concentrations were evaluated.
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, 55116 Mainz, Germany
- Type of assay:
- other: in vitro gene mutation study in mammalian cells
- Target gene:
- TK-locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: ATCC (Wesel, Germany)
- Cell doubling time (h): 10 - 12
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 medium supplemented with horse serum, sodium pyruvate and penicillin/streptomycin, 5.0 ±
0.5% CO2
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: 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 Aroclor 1254 for 5 days
- Test concentrations with justification for top dose:
- Pre-test: 663.0, 331.5, 165.8, 82.9, 41.4, 20.7, and 10.4 µg/mL
Experiment I (+S9): 665.0, 332.5, 166.3, 83.1, 41.6, and 20.8 µg/mL
Experiment I (-S9): 332.5, 166.3, 83.1, 41.6, 20.8, and 10.4 µg/mL
Experiment II (-S9): 166.4, 83.2, 41.6, 20.8, 10.4, and 5.2 µg/mL
Precipitation was noted at the highest test item concentration (665.0 μg/mL). Turbidity occurred down to a concentration of 166.3 μg/mL. Relevant cytotoxic effects were determined at 665.0 μg/mL following 4 h-treatment with metabolic activation and at 665.0 μg/mL and 332.5 μg/mL following 4 h-treatment without metabolic activation. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The test item was not soluble in any of the solvents usually employed. The best result was obtained with ethanol. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- methylmethanesulfonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding: approx. 1 x 10E7
SELECTION AGENT: trifluorothymidine (TFT), 5 µg/mL
NUMBER OF REPLICATIONS: two
EXPERIMENTAL PROCEDURE AND DETAILS:
Pre-Test for Cytotoxicity (with and without S9-mix, two replicates per culture, 7 concentrations):
- Treatment for 4 h in the presence and in the absence of S9
- Remove treatment medium (with/without S9-mix) and test item
- Add fresh medium after washing
- Transfer of 2 cells per well into 96-well microtiter plates
- Incubation for 10-12 days
- Evaluation of relative cloning efficiency
Experiment I with S9-mix (two replicates per culture, 6 concentrations):
- Treatment for 4 h in the presence of S9-mix
- Remove treatment medium, S9-mix and test item
- Add fresh medium after washing
- Measure cell density directly after treatment as well as 24 ± 4 h and 48 ± 4 h after treatment
- Evaluation of % relative suspension growth (RSG)
- Transfer of cells into 96-well microtiter plates (2 cells per well for viability and 4000 cells per well for mutagenicity (with TFT))
- Incubation for 10-12 days
- Evaluation of cytotoxicity and mutagenicity
Experiment I without S9-mix (two replicates per culture, 6 concentrations):
- Treatment for 4 h in the absence of S9-mix
- Remove treatment medium and test item
- Add fresh medium after washing
- Measure cell density directly after treatment as well as 24 ± 4 h and 48 ± 4 h after treatment
- Evaluation of % relative suspension growth (RSG)
- Transfer of cells into 96 well microtiter plates (2 cells per well for viability and 4000 cells per well for mutagenicity (with TFT))
- Incubation for 10-12 days
- Evaluation of cytotoxicity and mutagenicity
Experiment II without S9, extended exposure (two replicates per culture, 6 concentrations):
- Treatment for 24 h in the absence of S9
- Remove treatment medium and test item
- Add fresh medium after washing
- Measure cell density directly after treatment as well as 24 ± 4 h and 48 ± 4 h after treatment
- Evaluation of toxicity and % relative suspension growth (RSG)
- Transfer of cells into 96-well microtiter plates (2 cells per well for viability 4000 cells per well for mutagenicity (with TFT))
- Incubation for 10-12 days
- Evaluation of cytotoxicity and mutagenicity
DETERMINATION OF CYTOTOXICITY
- Method: relative cloning efficiency - Evaluation criteria:
- The test item is considered to have mutagenic effects if:
- the induced mutant frequency reproducibly exceeds a threshold of 126 colonies per 1 x 10E6 cells above the corresponding solvent control.
- the relative increase of the mutant frequency shows a dose relationship.
The test item is considered to have no mutagenic effects if:
- the induced mutation frequency does not exceeds a threshold of 126 colonies per 1 x 10E6 cells above the corresponding solvent control.
- the relative increase of the mutation frequency does not show a does relationship.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures. Results of test groups are generally rejected if the relative total growth is less than 10% of the solvent control. The biological relevance of the results was always considered first. Appropriate statistical methods were used as an aid in evaluating the test results. However, the results of statistical testing were assessed with respect to dose-response relationship. Reproducibility and historical data were also taken into consideration.
Reference: Moore MM, Honma M, Clements J, et al. (2007): Mouse lymphoma thymidine kinase gene mutation assay: meeting of the International Workshop on Genotoxicity Testing, San Francisco, 2005, recommendations for 24-h treatment. Mutat Res.627(1):36-40. - Statistics:
- Statistical significance was confirmed by means of the non-parametric χ2 test. However, both biological and statistical significance were considered together.
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Experiment I: 665.0, 332.5 μg/mL (+S9); 332.5, 166.3 µg/mL (-S9) Experiment II: 166.3, 83.1, 41.6 µg/mL (-S9)
- 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: Potential effects have been assessed in a preparative experiment. No effects were observed.
- Effects of osmolality: Potential effects have been assessed in a preparative experiment. No effects were observed.
- Water solubility: Test item was not soluble in water
- Precipitation: Observed at 665.0 μg/mL and turbidity occurred down to a concentration of 166.3 μg/mL.
VALIDATION AND COMPARISON WITH HISTORICAL CONTROL DATA.
- positive controls showed a distinct increase in induced total mutant colonies and exceeded the number of mutant colonies of more than 300 in comparison to the corresponding solvent control.
- In both experiments (with and without S9-mix) the range of the solvent controls was in the range of the historical data as well as in the range of the spontaneous mutant frequency of the L5178Y cells. - Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 07 Aug - 09 Oct 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- adopted in 2016
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- adopted in 2017
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, 55116 Mainz, Germany
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- not applicable
- Species / strain / cell type:
- lymphocytes: cultured peripheral lymphocytes treated with anti-coagulant (heparin)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Cell cycle length, doubling time or proliferation index: not specified
- Sex, age and number of blood donors if applicable: male and female healthy volunteers (approximately 18 - 35 years of age), non-smokers, with no known illness or recent exposure to genotoxic agents
- Whether whole blood or separated lymphocytes were used: whole blood
MEDIA USED
- Type and identity of media including CO2 concentration: RPMI 1640 medium (CO2 concentration 5 ± 0.5%) supplemented with
- 15%(v/v) fetal calf serum (FCS)
- 1% (v/v) penicillin/streptomycin (per mL: 10,000 IU Pen / 10 mg Strep)
- 2% (v/v) phytohaemagglutinin solution - Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9-mix), prepared from the livers of rats treated with Aroclor 1254
- Test concentrations with justification for top dose:
- 4 h-treatment with and without metabolic activation: 20.8, 41.6, 83.1, 166.3, 332.5 and 665.0 µg/mL
24 h-treatment without metabolic activation: 20.8, 41.6, 83.1, 166.3, 332.5 and 665.0 µg/mL
As the highest test item concentration (665.0 µg/mL) showed severe cytotoxicity, the 4 subsequent lower concentrations were selected for the evaluation of structural chromosome aberrations. - Vehicle / solvent:
- - Vehicles/solvents used: ethanol (0.5%)
- Justification for choice of solvent/vehicle: In a non-GLP pre-test, the solubility of the test item was determined in demineralized water, ethanol, tetrahydrofuran, acetone and dimethyl sulfoxide (DMSO). The test item was not soluble in any of the solvents. The best results were obtained with ethanol. At a concentration of 133 mg/mL and after 45 minutes ultrasonic treatment, a homogeneous and pipettable suspension could be achieved. However, even at this concentration a separation in two phases was visible after some hours. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 h with and without metabolic activation and 24 h without metabolic activation
SPINDLE INHIBITOR: 0.1 μg/mL colchicine (3 h before harvest)
STAIN: 10% solution of Giemsa
NUMBER OF REPLICATIONS: duplicates in two valid independent experiments (first approach without metabolic activation in experiment I was invalid (technical error) and had to be repeated)
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Each cell culture was harvested and processed separately. The cells were spun down by gentle centrifugation (10 min, 500 g). The supernatant was discarded and the cells were re-suspended in 12 mL hypotonic KCl solution. Then the cell suspension was allowed to stand at room temperature for 20 min. After removal of the hypotonic solution by centrifugation (10 min, 500 g), the cell pellet was exposed to fixative (3:1 mixture of methanol and glacial acetic acid). After fixation at 2 - 8 °C, minimum 30 min, the cell suspension was spun down by gentle centrifugation (10 min, 500 g), the supernatant was discarded and the cell pellet was re-suspended in fixative again. The washing procedures were repeated until the cell pellet was white. The slides were prepared by dropping the cell suspension onto clean microscope slides. The slides were then stained with a 10% solution of Giemsa. All slides were independently coded before microscopic analysis.
NUMBER OF CELLS EVALUATED: 1000
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: At least 300 well-spread metaphases
per concentration – 150 metaphases per replicate, if applicable - were scored for cytogenetic damage.
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Only metaphases with 46 ± 2 centromeric regions were included in the analysis.
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index - Evaluation criteria:
- A test item is classified as non-mutagenic if:
• Neither a statistically significant nor a concentration-related increase of the number of cells with structural chromosomal aberrations in the evaluated test concentrations is observed.
• The obtained results lie within the range of the historical laboratory control data for solvent controls.
A test item is classified as mutagenic if in any of the experimental conditions:
• The number of induced structural chromosome aberrations (excluding gaps) lies above the range of the historical laboratory control data for solvent controls, at least at one concentration of the test item.
• A dose-related increase in the number of cells with structural chromosome aberrations is observed.
• A statistically significant increase of structural chromosomal aberrations is found, at least at one concentration of the test item. - Statistics:
- The number of metaphases with structural aberrations in each treatment group was compared with the solvent control value. Statistical significance was tested using Fisher’s exact test at the 5% level (p < 0.05).
- Key result
- Species / strain:
- lymphocytes: cultured peripheral human lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- In both valid experiments without metabolic activation, cytotoxicity in varying degrees was detected. In the approach with metabolic activation, no cytotoxic effect was detected.
- 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: None of the tested positive and solvent controls or the test item provoked a critical change of the pH value in comparison to the solvent controls.
- Effects of osmolality: None of the tested positive and solvent controls or the test item provoked a critical change of the osmolarity in comparison to the solvent controls.
- Precipitation: Precipitation of the test substance was not observed.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
see Tables 1 and 2 - Conclusions:
- Interpretation of results: negative
Referenceopen allclose all
Table 1: Mean Revertants Experiment 1a (all strains tested)
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
||||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
Demin. water |
Mean |
85 |
80 |
29 |
34 |
73 |
93 |
307 |
377 |
27 |
26 |
sd |
6.9 |
12.7 |
6.4 |
9.8 |
5.9 |
13.6 |
48.4 |
61.1 |
1.5 |
7.0 |
|
DMSO |
Mean |
98 |
97 |
35 |
38 |
78 |
78 |
320 |
388 |
22 |
24 |
sd |
19.6 |
4.0 |
5.9 |
6.7 |
5.5 |
3.5 |
31.2 |
28.8 |
3.2 |
4.0 |
|
Ethanol |
Mean |
76 |
83 |
24 |
26 |
72 |
72 |
295 |
313 |
22 |
22 |
sd |
11.4 |
7.0 |
6.4 |
6.0 |
10.6 |
8.6 |
96.4 |
37.2 |
3.8 |
5.9 |
|
Positive Controls* |
Mean |
637 |
781 |
383 |
86 |
451 |
1528 |
1411 |
1472 |
323 |
183 |
sd |
99.4 |
64.2 |
27.2 |
8.1 |
22.7 |
424.2 |
100.0 |
36.7 |
56.6 |
28.4 |
|
f(I) |
6.50 |
8.05 |
10.94 |
2.26 |
6.18 |
19.59 |
4.41 |
3.79 |
11.96 |
7.63 |
|
5000 µg/plate |
Mean |
99 |
93 |
22 |
26 |
76 |
82 |
79 |
48 |
20 |
19 |
sd |
18.9 |
15.1 |
3.1 |
5.9 |
4.0 |
18.9 |
12.6 |
14.5 |
2.1 |
1.5 |
|
f(I) |
1.30 |
1.12 |
0.92 |
1.00 |
1.06 |
1.14 |
0.27 |
0.15 |
0.91 |
0.86 |
|
1500 µg/plate |
Mean |
79 |
98 |
24 |
21 |
75 |
74 |
66 |
95 |
17 |
19 |
sd |
16.2 |
9.0 |
4.0 |
6.4 |
11.5 |
8.0 |
19.9 |
23.1 |
1.5 |
2.5 |
|
f(I) |
1.04 |
1.18 |
1.00 |
0.81 |
1.04 |
1.03 |
0.22 |
0.30 |
0.77 |
0.86 |
|
500 µg/plate |
Mean |
90 |
94 |
26 |
24 |
82 |
77 |
285 |
331 |
23 |
19 |
sd |
22.0 |
15.9 |
5.5 |
2.9 |
6.0 |
13.6 |
60.2 |
10.1 |
4.5 |
3.2 |
|
f(I) |
1.18 |
1.13 |
1.08 |
0.92 |
1.14 |
1.07 |
0.97 |
1.06 |
1.05 |
0.86 |
|
150 µg/plate |
Mean |
79 |
84 |
26 |
20 |
66 |
76 |
281 |
384 |
18 |
23 |
sd |
15.0 |
20.3 |
6.9 |
2.6 |
5.5 |
10.7 |
89.3 |
10.6 |
3.6 |
0.6 |
|
f(I) |
1.04 |
1.01 |
1.08 |
0.77 |
0.92 |
1.06 |
0.95 |
1.23 |
0.82 |
1.05 |
|
50 µg/plate |
Mean |
88 |
89 |
25 |
32 |
83 |
64 |
291 |
355 |
22 |
24 |
sd |
5.2 |
6.4 |
4.0 |
1.2 |
14.5 |
6.2 |
63.8 |
64.7 |
4.6 |
4.2 |
|
f(I) |
1.16 |
1.07 |
1.04 |
1.23 |
1.15 |
0.89 |
0.99 |
1.13 |
1.00 |
1.09 |
|
f(I) = increase factor
* Different positive controls were used
Table 2: Mean Revertants Experiment 1b (strain TA 102)
Strain |
TA102 |
||
Induction |
-S9 |
+S9 |
|
Demin. water |
Mean |
356 |
345 |
sd |
62.9 |
72.6 |
|
DMSO |
Mean |
257 |
324 |
sd |
26.6 |
20.8 |
|
Ethanol |
Mean |
292 |
258 |
sd |
58.9 |
24.0 |
|
Positive Controls* |
Mean |
689 |
1083 |
sd |
60.2 |
400.0 |
|
f(I) |
2.68 |
3.34 |
|
5000 µg/plate |
Mean |
12 |
4 |
sd |
1.0 |
1.5 |
|
f(I) |
0.04 |
0.02 |
|
1500 µg/plate |
Mean |
80 |
56 |
sd |
10.0 |
9.0 |
|
f(I) |
0.27 |
0.22 |
|
500 µg/plate |
Mean |
315 |
268 |
sd |
46.4 |
58.9 |
|
f(I) |
1.08 |
1.04 |
|
150 µg/plate |
Mean |
284 |
284 |
sd |
52.3 |
52.0 |
|
f(I) |
0.97 |
1.10 |
|
50 µg/plate |
Mean |
355 |
292 |
sd |
50.0 |
34.6 |
|
f(I) |
1.22 |
1.13 |
|
15 µg/plate |
Mean |
339 |
349 |
sd |
47.7 |
120.2 |
|
f(I) |
1.16 |
1.35 |
|
5 µg/plate |
Mean |
304 |
287 |
sd |
65.8 |
33.5 |
|
f(I) |
1.04 |
1.11 |
|
f(I) = increase factor
* Different positive controls were used
Table 3: Mean Revertants Experiment 2 (strains TA 97a, TA 98, TA 100 and TA 1535)
Strain |
TA97a |
TA98 |
TA100 |
TA1535 |
|||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
Demin. water |
Mean |
64 |
81 |
24 |
28 |
51 |
64 |
22 |
23 |
sd |
4.0 |
17.9 |
4.7 |
2.1 |
4.0 |
6.7 |
4.2 |
6.1 |
|
DMSO |
Mean |
82 |
67 |
28 |
34 |
44 |
62 |
24 |
24 |
sd |
13.7 |
11.0 |
8.2 |
5.6 |
3.6 |
7.5 |
4.4 |
4.2 |
|
Ethanol |
Mean |
61 |
87 |
32 |
35 |
40 |
48 |
21 |
23 |
sd |
8.5 |
14.2 |
4.0 |
4.2 |
1.0 |
1.5 |
5.0 |
4.9 |
|
Positive Controls* |
Mean |
631 |
237 |
459 |
165 |
429 |
491 |
280 |
141 |
sd |
83.2 |
59.8 |
88.1 |
8.1 |
113.5 |
132.8 |
59.7 |
9.2 |
|
f(I) |
7.70 |
3.54 |
16.39 |
4.85 |
8.41 |
7.92 |
12.73 |
5.88 |
|
5000 µg/plate |
Mean |
71 |
80 |
22 |
29 |
54 |
64 |
15 |
17 |
sd |
5.7 |
1.2 |
7.2 |
3.0 |
6.1 |
8.5 |
1.7 |
2.6 |
|
f(I) |
1.16 |
0.92 |
0.69 |
0.83 |
1.35 |
1.33 |
0.71 |
0.74 |
|
2500 µg/plate |
Mean |
69 |
76 |
25 |
29 |
46 |
73 |
18 |
26 |
sd |
2.5 |
5.0 |
4.9 |
1.5 |
9.1 |
6.1 |
2.0 |
2.5 |
|
f(I) |
1.13 |
0.87 |
0.78 |
0.83 |
1.15 |
1.52 |
0.86 |
1.13 |
|
1250 µg/plate |
Mean |
86 |
103 |
21 |
31 |
52 |
63 |
18 |
23 |
sd |
14.7 |
19.3 |
2.1 |
10.1 |
7.0 |
14.5 |
2.0 |
5.5 |
|
f(I) |
1.41 |
1.18 |
0.66 |
0.89 |
1.30 |
1.31 |
0.86 |
1.00 |
|
625 µg/plate |
Mean |
71 |
85 |
26 |
28 |
57 |
68 |
18 |
28 |
sd |
12.7 |
17.2 |
9.6 |
4.7 |
4.6 |
9.0 |
1.5 |
3.5 |
|
f(I) |
1.16 |
0.98 |
0.81 |
0.80 |
1.43 |
1.42 |
0.86 |
1.22 |
|
313 µg/plate |
Mean |
66 |
72 |
28 |
25 |
42 |
56 |
17 |
23 |
sd |
0.0 |
4.2 |
5.2 |
4.5 |
7.0 |
5.6 |
2.3 |
2.1 |
|
f(I) |
1.08 |
0.83 |
0.88 |
0.71 |
1.05 |
1.17 |
0.81 |
1.00 |
|
156 µg/plate |
Mean |
74 |
95 |
30 |
35 |
55 |
69 |
21 |
26 |
sd |
2.9 |
4.0 |
7.2 |
7.9 |
8.7 |
8.1 |
6.6 |
4.0 |
|
f(I) |
1.21 |
1.09 |
0.94 |
1.00 |
1.38 |
1.44 |
1.00 |
1.13 |
|
78 µg/plate |
Mean |
72 |
74 |
27 |
28 |
37 |
45 |
16 |
23 |
sd |
4.6 |
6.4 |
8.0 |
9.0 |
7.9 |
11.4 |
3.1 |
2.9 |
|
f(I) |
1.18 |
0.85 |
0.84 |
0.80 |
0.93 |
0.94 |
0.76 |
1.00 |
|
f(I) = increase factor
* Different positive controls were used
Table 4: Mean Revertants Experiment 2 (strain TA 102)
Strain |
TA102 |
||
Induction |
-S9 |
+S9 |
|
Demin. water |
Mean |
287 |
283 |
sd |
19.7 |
28.1 |
|
DMSO |
Mean |
287 |
268 |
sd |
42.0 |
38.2 |
|
Ethanol |
Mean |
253 |
335 |
sd |
42.4 |
43.9 |
|
Positive Controls* |
Mean |
952 |
1325 |
sd |
298.2 |
51.4 |
|
f(I) |
3.32 |
4.94 |
|
500 µg/plate |
Mean |
320 |
332 |
sd |
39.4 |
35.6 |
|
f(I) |
1.26 |
0.99 |
|
250 µg/plate |
Mean |
292 |
292 |
sd |
13.9 |
10.6 |
|
f(I) |
1.15 |
0.87 |
|
125 µg/plate |
Mean |
301 |
287 |
sd |
58.0 |
26.6 |
|
f(I) |
1.19 |
0.86 |
|
62.5 µg/plate |
Mean |
271 |
269 |
sd |
24.1 |
64.8 |
|
f(I) |
1.07 |
0.80 |
|
31.3 µg/plate |
Mean |
276 |
283 |
sd |
52.9 |
42.0 |
|
f(I) |
1.09 |
0.84 |
|
15.6 µg/plate |
Mean |
289 |
315 |
sd |
42.8 |
77.7 |
|
f(I) |
1.14 |
0.94 |
|
7.8 µg/plate |
Mean |
280 |
275 |
sd |
17.4 |
15.1 |
|
f(I) |
1.11 |
0.82 |
|
f(I) = increase factor
* Different positive controls were used
Table 5: Historical Data of Spontaneous Revertants and Comparison with Experiments 1a, 1b and 2
Strain |
|
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
|||||
Induction |
|
- S9 |
+ S9 |
- S9 |
+ S9 |
- S9 |
+ S9 |
- S9 |
+ S9 |
- S9 |
+ S9 |
H2O de-min. |
Mean |
92 |
97 |
15 |
18 |
93 |
97 |
279 |
296 |
17 |
17 |
Min |
60 |
63 |
6 |
8 |
62 |
66 |
85 |
67 |
6 |
7 |
|
Max |
144 |
138 |
37 |
41 |
141 |
141 |
425 |
511 |
31 |
33 |
|
SD |
19 |
16 |
6 |
6 |
15 |
15 |
65 |
75 |
6 |
6 |
|
Exp 1a |
85 |
80 |
29 |
34 |
73 |
93 |
307 |
377 |
27 |
26 |
|
Exp 1b |
n.r. |
n.r. |
n.r. |
n.r. |
n.r. |
n.r. |
356 |
345 |
n.r. |
n.r. |
|
Exp 2 |
64 |
81 |
24 |
28 |
51 |
64 |
287 |
283 |
22 |
23 |
|
DMSO |
Mean |
91 |
100 |
15 |
16 |
90 |
93 |
279 |
289 |
17 |
16 |
Min |
58 |
70 |
7 |
8 |
60 |
63 |
79 |
80 |
8 |
6 |
|
Max |
135 |
144 |
46 |
36 |
136 |
199 |
393 |
459 |
33 |
31 |
|
SD |
18 |
16 |
7 |
6 |
16 |
19 |
61 |
65 |
6 |
6 |
|
Exp 1a |
98 |
97 |
35 |
38 |
78 |
78 |
320 |
388 |
22 |
24 |
|
Exp 1b |
n.r. |
n.r. |
n.r. |
n.r. |
n.r. |
n.r. |
257 |
324 |
n.r. |
n.r. |
|
Exp 2 |
82 |
67 |
28 |
34 |
44 |
62 |
287 |
268 |
24 |
24 |
|
Ethanol |
Mean |
89 |
99 |
16 |
17 |
86 |
90 |
284 |
275 |
16 |
16 |
Min |
57 |
65 |
8 |
9 |
61 |
69 |
111 |
141 |
9 |
9 |
|
Max |
181 |
205 |
35 |
40 |
116 |
129 |
368 |
339 |
28 |
29 |
|
SD |
24 |
29 |
7 |
8 |
13 |
16 |
52 |
47 |
6 |
6 |
|
Exp 1a |
76 |
83 |
24 |
26 |
72 |
72 |
295 |
313 |
22 |
22 |
|
Exp 1b |
n.r. |
n.r. |
n.r. |
n.r. |
n.r. |
n.r. |
292 |
258 |
n.r. |
n.r. |
|
Exp 2 |
61 |
87 |
32 |
35 |
40 |
48 |
253 |
335 |
21 |
23 |
|
Positive Controls* |
Mean |
552 |
501 |
394 |
83 |
513 |
716 |
1160 |
1237 |
254 |
115 |
Min |
264 |
241 |
100 |
39 |
223 |
273 |
491 |
408 |
55 |
45 |
|
Max |
1152 |
1181 |
793 |
333 |
984 |
1912 |
2331 |
6083 |
484 |
712 |
|
SD |
169 |
144 |
143 |
49 |
153 |
290 |
452 |
659 |
89 |
79 |
|
Exp 1a |
637 |
781 |
383 |
86 |
451 |
1528 |
1411 |
1472 |
323 |
183 |
|
Exp 1b |
n.r. |
n.r. |
n.r. |
n.r. |
n.r. |
n.r. |
689 |
1083 |
n.r. |
n.r. |
|
Exp 2 |
631 |
237 |
459 |
165 |
429 |
491 |
952 |
1325 |
280 |
141 |
|
* Different positive controls were used
Table 2: Final results from experiment I and II (mean values of both cultures)
Content |
Relative Total Growth [%] |
Mutants per 106Cells |
||||
|
Exp I |
Exp II |
Exp I |
Exp II |
||
|
+S9 |
-S9 |
-S9 |
+S9 |
-S9 |
-S9 |
Threshold |
- |
- |
- |
182 |
178 |
176 |
Solvent Control of Test Item |
- |
- |
- |
56 |
52 |
50 |
Solvent Control of Positive Control |
- |
- |
- |
63 |
52 |
53 |
Positive Control |
54 |
54 |
32 |
378 |
358 |
441 |
Test Item 665.0 µg/mL |
0 |
|
|
* |
|
|
Test Item 332.5 µg/mL |
10 |
0 |
|
* |
* |
|
Test Item 166.3 µg/mL |
32 |
15 |
0 |
76 |
* |
* |
Test Item 83.1 µg/mL |
69 |
56 |
1 |
62 |
61 |
* |
Test Item 41.6 µg/mL |
82 |
93 |
10 |
56 |
55 |
* |
Test Item 20.8 µg/mL |
84 |
97 |
34 |
57 |
68 |
64 |
Test Item 10.4 µg/mL |
|
99 |
55 |
|
64 |
68 |
Test Item 5.2 µg/mL |
|
|
84 |
|
|
60 |
* Cultures were not evaluated because of cytotoxicity.
Note: The threshold is defined as the number of mutant colonies per 1 x 10E6 cells of the respective solvent control plus 126 and was defined by Moore et al., 2007
Moore MM, Honma M, Clements J, et al. (2007): Mouse lymphoma thymidine kinase gene mutation assay: meeting of the International Workshop on Genotoxicity Testing, San Francisco, 2005, recommendations for 24-h treatment. MutatRes.627(1):36-40.
Table 3. Results of Valid Cytotoxicity Experiment I without Metabolic Activation
Treatment |
Mitotic Index in % |
Mitotic Index in % of solvent control |
Cytotoxicity in % |
Solvent control MCM |
26.5 |
100 |
- |
Solvent control ethanol 0.5% v/v |
20.1 |
100 |
- |
Positive control EMS 300 µg/mL |
19.4 |
97 |
3 |
Positive control EMS 600 µg/mL |
15.9 |
79 |
21 |
Test item 665 µg/mL |
18.0 |
68 |
32 |
Test item 332.5 µg/mL |
11.9 |
45 |
55 |
Test item 166.3 µg/mL |
15.9 |
60 |
40 |
Test item 83.1 µg/mL |
21.5 |
81 |
19 |
Test item 41.6 µg/mL |
24.4 |
92 |
8 |
Test item 20.8 µg/mL |
27.4 |
104 |
-4 |
Generally, cytotoxicity was not a limiting factor for the evaluation of chromosomal aberrations, but the highest test item concentration did not show the highest cytotoxicity. Therefore, the 4 highest test item concentrations (including the concentration with the highest cytotoxicity of 55% and concentrations with moderate/low cytotoxicity) were scored for structural chromosomal aberrations.
Table 4. Results of Valid Cytotoxicity Experiment I with Metabolic Activation
Treatment |
Mitotic Index in % |
Mitotic Index in % of solvent control |
Cytotoxicity in % |
Solvent control ethanol 0.5% v/v |
12.1 |
100 |
- |
Solvent control 0.9% NaCl 0.5% v/v |
11.8 |
100 |
- |
Positive control CPA 30 µg/mL |
11.2 |
94 |
6 |
Positive control CPA 20 µg/mL |
10.7 |
90 |
10 |
Test item 665 µg/mL |
8.3 |
68 |
32 |
Test item 332.5 µg/mL |
9.2 |
76 |
24 |
Test item 166.3 µg/mL |
12.9 |
107 |
-7 |
Test item 83.1 µg/mL |
13.6 |
113 |
-13 |
Test item 41.6 µg/mL |
14.6 |
121 |
-21 |
Test item 20.8 µg/mL |
13.7 |
114 |
-14 |
No limiting cytotoxicity was observed. Therefore, the 3 highest test item concentrations were scored for structural chromosomal aberrations.
Table 5. Results Chromosomal Aberration Assay Experiment I - without Metabolic Activation
Treatment |
Aberrant cells in % |
||
Including gaps* |
Excluding gaps* |
with exchanges |
|
Without metabolic activation |
|||
Solvent control MCM |
0.7 |
0.7 |
0.0 |
Solvent control ethanol 0.5% v/v |
6.7 |
2.7 |
0.0 |
Positive control EMS 600 µg/ml |
21.0 |
16.7S |
4.0 |
Test item 665 µg/mL |
4.7 |
1.7 |
0.0 |
Test item 332.5 µg/mL |
3.6 |
1.6 |
0.0 |
Test item 166.3 µg/mL |
4.5 |
1.9 |
0.0 |
Test item 83.1 µg/mL |
3.3 |
1.0 |
0.0 |
With metabolic activation |
|||
Solvent control ethanol 0.5% v/v |
6.0 |
2.7 |
0.7 |
Solvent control 0.9% NaCl 0.5% v/v |
4.7 |
2.7 |
0.3 |
Positive control CPA 30 µg/ml |
20.7 |
14.0S |
2.0 |
Test item 665 µg/mL |
5.7 |
1.3 |
0.0 |
Test item 332.5 µg/mL |
7.0 |
2.0 |
0.5 |
Test item 166.3 µg/mL |
3.0 |
1.7 |
0.0 |
* including cells carrying exchanges
S aberration frequency statistically significantly higher than corresponding control values
Table 6. Results Cytotoxicity Experiment II without Metabolic Activation
Treatment |
Mitotic Index in % |
Mitotic Index in % of solvent control |
Cytotoxicity in % |
Solvent control MCM |
9.2 |
100 |
-- |
Solvent control ethanol 0.5% v/v |
9.5 |
100 |
-- |
Positive control EMS 300 µg/mL |
4.4 |
46 |
54 |
Positive control EMS 600 µg/mL |
1.6 |
16 |
84 |
Test item 665 µg/mL |
2.6 |
28 |
72 |
Test item 332.5 µg/mL |
8.1 |
89 |
11 |
Test item 166.3 µg/mL |
9.0 |
98 |
2 |
Test item 83.1 µg/mL |
10.1 |
110 |
-10 |
Test item 41.6 µg/mL |
9.4 |
102 |
-2 |
Test item 20.8 µg/mL |
9.6 |
104 |
-4 |
Table 7. Results Chromosomal Aberration Assay Experiment II
Treatment |
Aberrant cells in % |
||
Including gaps* |
Excluding gaps* |
with exchanges |
|
Solvent control MCM |
8.3 |
3.3 |
0 |
Solvent control ethanol 0.5% v/v |
6.3 |
2.0 |
0.3 |
Positive control EMS 300 mg/mL |
29.3 |
16.3S |
3.3 |
Test item 332.5 µg/mL |
6.0 |
2.0 |
0 |
Test item 166.3 µg/mL |
4.7 |
1.7 |
0 |
Test item 83.1 µg/mL |
7.0 |
2.3 |
0 |
Test item 41.6 µg/mL |
6.3 |
2.3 |
0 |
* including cells carrying exchanges
S aberration frequency statistically significantly higher than corresponding control values
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
In vitro gene mutation in bacteria and mammalian cells as well as chromosome aberration in mammalian cells were tested with 4-(octadecylamino)-4-oxoisocrotonic acid.
Genetic toxicity (mutagenicity) in bacteria in vitro
A bacterial gene mutation assay (Ames test) was performed with 4-(octadecylamino)-4-oxoisocrotonic acid according to OECD 471 (LAUS, 2017c). S. typhimurium strains TA 97a, TA 98, TA 100 and TA 102 and TA1535 were tested conducting both the plate incorporation and the pre-incubation method in the absence and presence of a metabolic activation (Aroclor 1254-induced rat liver S9-mix). The experiment was conducted in 3 replicates in two independent experiments each up to the limit concentration of 5000 µg/plate. Cytotoxicity was observed for S. typhimurium strain TA102 at 5000 and 1500 µg/plate (with and without metabolic activation) in the plate incorporation test, therefore the dose used for the pre-incubation method was reduced to a maximum of 500 µg/plate. No increase in the number of revertant colonies was noted in any of the bacterial strains, with and without metabolic activation system. Appropriate positive and solvent controls were included in the test and showed the expected results. Under the conditions of the study, 4-(octadecylamino)-4-oxoisocrotonic acid did not induce mutations in the bacterial mutation assay in the absence and presence of a metabolic activation system in any of the strains tested.
Genetic toxicity (cytogenicity) in mammalian cells in vitro
An in vitro mammalian chromosome aberration test was conducted with 4-(octadecylamino)-4-oxoisocrotonic acid in accordance with OECD 473 under GLP conditions (LAUS, 2018b). The induction of structural chromosome aberrations was evaluated in vitro in lymphocytes of cultured heparinised human whole blood, incubated for 4 h or 24 h with and without metabolic activation (S9-mix from rats treated with Arocolor 1254). Test substance concentrations of 20.8 - 665 µg/mL with and without metabolic activation were applied. The negative as well as the positive controls showed the expected results. No statistically or biologically significant increase in the incidence of chromosome aberrations was observed. Therefore, under the conditions of the study, the test substance did not show clastogenic activity in this chromosomal aberration test with and without metabolic activation performed in peripheral human lymphocytes in vitro.
Genetic toxicity (mutagenicity) in mammalian cells in vitro
The in vitro mammalian cell gene mutation study of 4-(octadecylamino)-4-oxoisocrotonic acid was carried out according to OECD 490 under GLP conditions (LAUS, 2018c). Gene mutations in the thymidine kinase locus were investigated in L5178Y mouse lymphoma cells in the presence and absence of a metabolic activation system (Arocolor 1254-induced rat liver, S9-mix). In the first experiment, cells were exposed to six test substance concentrations between 20.8 - 665.0 µg/mL with metabolic activation and six concentrations between 10.4 - 332.5 µg/mL without metabolic activation for 4 h. In the second experiment, cells were exposed to six test substance concentrations between 5.2 -166.4 µg/mL without metabolic activation for 24 h. The vehicle and positive controls in the study showed the expected results and were within the range of historical control data of the laboratory. Precipitation was noted at the highest test substance concentration (665.0 μg/mL). Turbidity occurred down to a concentration of 166.3 μg/mL. Relevant cytotoxic effects were determined in the first experiment at 665.0 μg/mL following 4 h treatment with metabolic activation and at 665.0 μg/mL and 332.5 μg/mL following 4 h treatment without metabolic activation. In the second experiment without metabolic activation a cytotoxic effect was observed at concentrations of 166.3 μg/mL, 83.1 μg/mL and 41.6 μg/mL. These concentrations were not evaluated for mutagenicity. There was no significant increase in the number of forward mutations at the thymidine kinase locus of L5178Y mouse lymphoma cells treated with the test subtsance with and without metabolic activation. Under the conditions of the study, 4-(octadecylamino)-4-oxoisocrotonic acid did not show gene mutation activity in this test performed in L5178Y mouse lymphoma cells in vitro.
Conclusion
Taken together, the available data on genetic toxicity of 4-(octadecylamino)-4-oxoisocrotonic acid do not indicate any mutagenic and clastogenic potential in vitro.
Justification for classification or non-classification
The available data on genetic toxicity of the registered substance do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 and are, therefore, conclusive but not sufficient for classification.
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