Registration Dossier
Registration Dossier
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EC number: 921-910-2 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
- Bacterial mutagenicity: Not mutagenic in the Ames test with and without metabolic activation
- Mammalian cytotoxicity: Not mutagenic in the in-vitro micronucleus test
- Mammalian genotoxicity: Not mutagenic in a forward mutation test (Mouse Lymphoma cells)
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2017
- 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)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 16/0437-1, 389-48
- Expiration date of the lot/batch: 2019-05-23
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature - Species / strain / cell type:
- lymphocytes: human lymphocytes
- Details on mammalian cell type (if applicable):
- blood samples were drawn from healthy non-smoking donors not receiving medication.
- Metabolic activation:
- with and without
- Metabolic activation system:
- Mammalian Microsomal Fraction S9 mix
- Test concentrations with justification for top dose:
- 1st Run: 3.0, 5.3, 9.2, 16.1, 28.2, 49.4, 86.5, 151, 265, 795, 2384µg/plate
2nd Run: 4.2, 7.3, 12.8, 22.4, 39.2, 68.6, 120, 300µg/plate
With regard to the purity (83.9%) and the solubility properties of the test item, 2384 μg/mL were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 3.0 to 2384 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. Clear toxic effects were observed after 4 hours treatment only with 795 μg/mL in the presence of S9 mix. Considering the precipitation data of Experiment I, 300 μg/mL (without S9 mix) were chosen as top concentration in Experiment II. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: Demecolcin
- Details on test system and experimental conditions:
- NUMBER OF CELLS EVALUATED: 1000 binucleate cells
- Evaluation criteria:
- The micronucleus assay will be considered acceptable if it meets the following criteria:
− The concurrent solvent control will normally be within the laboratory historical solvent control data range (95% control limit realized as 95% confidence interval).
− The concurrent positive controls should produce a statistically significant increase in the micronucleus frequency and should be within the laboratory historical positive control data range.
− Cell proliferation criteria in the solvent control are considered to be acceptable.
− All experimental conditions described earlier were tested unless one exposure condition resulted in a clearly positive result.
− The quality of the slides must allow the evaluation of an adequate number of cells and concentrations. - Statistics:
- Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.
- Species / strain:
- lymphocytes: Human lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no relevant effect observed
- Effects of osmolality: no relevant effect observed
- Precipitation: observed at 49.4 µg/ml and above in the absence of S9 mix and at 86.5µg/ml and above in the presence of S9 mix at the end of treatment. - Conclusions:
- In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to precipitating concentrations. - Executive summary:
The test item, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments. In each experimental group two parallel cultures were analyzed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage. The highest applied concentration in this study (2384 μg/mL of the test item) was chosen with regard to the purity (83.9%) and the solubility properties of the test item and with respect to the current OECD Guideline 487. Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 487. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentrations, which showed precipitation. In the absence and presence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to precipitating concentrations.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 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)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- histidine and tryptophan
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose was tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 μL/plate might also be tested in repeat experiments for further clarification/substantiation.
In this study, due to the purity of the test substance 5.8 mg/plate was used as top dose in all experiments. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available. - Untreated negative controls:
- other:
- Remarks:
- sterility control
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- other: 2-aminoanthracene, n-methyl-N`-nitro-N-nitrosoguanidine
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48-72h at 37°C in the dark
NUMBER OF REPLICATIONS: 3 - Evaluation criteria:
- Generally, the experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 10^9 cells per mL were used.
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other. - Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- In the SPT from about 2900µg/plate in the presence of S9 mix
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- In the SPT from about 5800µg/plate in the presence and absence of S9 mix and in the PIT from about 2900µg/plate in the presence and absence of S9 mix.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- In the SPT from about 2900µg/plate in the presence of S9 mix and in the PIT from about 5800µg/plate in the presence and absence of S9 mix.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- In the SPT from about 5800µg/plate in the absence of S9 mix and in the PIT from about 5800µg/plate in the absence and presence of S9 mix.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- In the SPT and PIT from about 2900µg/plate in the absence of S9 mix and from about 5800µg/plate in the presence of S9 mix.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Precipitation of the test substance was found from about 333 μg/plate onward in the standard plate test and from about 1000 μg/plate onward in the preincubation test with and without S9 mix.
- Conclusions:
- Under the experimental conditions chosen here, it is concluded that the test item is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.
- Executive summary:
The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay (TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA) in a dose range of 33 μg - 5800 μg/plate (SPT) and 33 μg - 5800 μg/plate (PIT). The Standard plate test (SPT) and preincubation test (PIT) were performed both with and without metabolic activation (liver S9 mix from induced rats). Precipitation of the test substance was found from about 333 μg/plate onward in the standard plate test and from about 1000 μg/plate onward in the preincubation test with and without S9 mix. A bacteriotoxic effect was observed depending on the strain and test conditions from about 2900 μg/plate onward. A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.
Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 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)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: In vitro gene mutation test in L5178Y mouse lymphoma cells
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 16/0437-1, 389-48
- Expiration date of the lot/batch:2019-05-23
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature - Target gene:
- thymidine kinase
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- The concentrations tested in this study were selected in accordance with the requirements set forth in the test guidelines and based on the results of a preliminary range finding test.
1st Run: 6.25, 12.50, 25.00, 50.00, 100.00, 200.00µg/ml (+S9/-S9)
2nd Run: 6.25, 12.50, 25.00, 50.00, 100.00, 200.00µg/ml (-S9)
4.69, 9.38, 18.75, 37.50, 75.00, 150.00µg/ml (+S9) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test substance was soluble in DMSO. - Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- cyclophosphamide
- methylmethanesulfonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 3 x 10^5 cells per 75 cm² flask in a total volume of 30 ml.
SELECTION AGENT (mutation assays):
During the week prior to treatment, spontaneous TK deficient mutants (TK-/-) were eliminated from the stock cultures by incubating 3 x 105 cells per 75 cm² flask in a total volume of 30 mL for 1 day in “THMG" medium (pretreatment medium A), and for the following 3 days in “THG" medium (pretreatment medium B).
For the selection of the mutants, the cells were centrifuged (173 g, 5 min) and 5 x 105 cells from each culture were resuspended in 50 mL selection medium (“TFT" medium; 1 x 104 cells/mL). Per culture 200 μL were dispensed in each well of two 96-well plates (2000 cells/well). After incubation for at least 9 days, both the number of negative wells and the number of wells containing small or large colonies were scored for calculation of the mutant frequency (MF).
Cloning efficiency 1 (survival)
At the end of the exposure period, the cells were centrifuged (173 g, 5 min) and 400 cells from each test group were resuspended in 50 mL RPMI-20 medium (8 cells/mL). Per culture 200 μL were dispensed in each well of two 96-well plates (1.6 cells/well). After incubation for 9 - 11 days the plates were scored for empty wells.
Cloning efficiency 2 (viability)
After the expression period, 2 days after end of exposure, the cells were centrifuged (173 g, 5 min) and 400 cells from each culture were resuspended in 50 mL RPMI-20 medium (8 cells/mL). Per culture 200 μL were dispensed in each well of two 96-well plates (1.6 cells/well). After incubation for at least 9 days the plates were scored for empty wells.
Relative suspension growth and relative total growth
For calculation of the relative suspension growth (RSG) and the relative total growth (RTG) the cell counts determined within the expression period at 2nd and 3rd passage after exposure in the case of 4-hour exposure and 1st, 2nd and 3rd passage after exposure in the case of 24-hour exposure were used. In the pretest single cultures per test group were conducted only.
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:
- Any supplementary information relevant to cytotoxicity: - Evaluation criteria:
- The MLTK assay is considered valid if the following criteria are met considering the international guidelines and the current recommendations of the IWGT (7, 9, 10, 11, 12):
- The absolute cloning efficiency obtained at the time of mutant selection (CE2) of the negative/vehicle controls should fall in the range of 65 - 120%.
- The SG of the negative/vehicle controls referring to the expression period following treatment should fall in the range of 8 - 32 for 4-hour exposure and 32 - 180 for 24-hour exposure.
- The mutant frequency of the negative/vehicle controls should fall within the range of 50 - 170 x 10-6 colonies.
- The positive controls should yield an absolute increase in total MF that is an increase above the spontaneous background MF (an induced MF [IMF]) of at least 300 x 10-6 colonies. The small colony MF should account for at least 40% of that IMF, means a small colony IMF of at least 120 x 10-6 colonies. Alternatively, the positive controls should induce at least 150 x 10-6 small colonies above the spontaneous background MF. The upper limit of cytotoxicity observed in the positive controls should have a RTG that is greater than 10%.
- The highest applied concentration of the test substance should be 5 mg/mL, 5 μL/mL or 10 mM, unless limited by cytotoxicity or solubility of the test substance. If toxicity occurs, the highest concentration should lower the RTG to 10 to 20% of survival. If precipitation
occurs, the highest evaluated concentration should be the lowest concentration where precipitation is observed by the unaided eye. - Statistics:
- An appropriate statistical method to test for linear trend (MS EXCEL function RGP; 10) was performed to assess a possible linear dose-relation in mutant frequencies. The dependent variable was the corrected mutant frequency and the independent variable was the
concentration. A trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05 and the slope was greater than 0. However, both, biological and statistical significance have been considered together. - Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Thus, under the experimental conditions chosen here, the conclusion is drawn that the test item does not induce forward mutations or chromosomal aberrations in vitro in the mouse lymphoma assay with L5178Y TK+/- cells in the absence and the presence of metabolic activation.
- Executive summary:
The test substance was tested for its ability to induce gene mutations at the thymidine kinase (TK) locus or structural chromosome aberrations at chromosome 11 in L5178Y TK+/- mouse lymphoma cells in vitro with the microwell method. Two independent experiments were carried out with and without the addition of liver S9 mix from phenobarbital and -naphthoflavone induced rats (exogenous metabolic activation).
According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following concentrations were tested and evaluated for gene mutations.
1st Experiment
without S9 mix (4-hour exposure period)
0; 6.25; 12.50; 25.00; 50.00; 100.00; 200.00 μg/mL
with S9 mix (4-hour exposure period)
0; 6.25; 12.50; 25.00; 50.00; 100.00; 200.00 μg/mL
2nd Experiment
without S9 mix (24-hour exposure period)
0; 6.25; 12.50; 25.00; 50.00; 100.00; 200.00 μg/mL
with S9 mix (4-hour exposure period)
0; 4.69; 9.38; 18.75; 37.50; 75.00; 150.00 μg/mL
Cells were treated with the test substance for 4 and 24 hours in the absence of metabolic activation and for 4 hours in the presence of metabolic activation. Subsequently, cells were cultured for an expression period of about 48 hours and then cultured in selection medium for another approx. 10 days. Finally, the number of large and small colonies was determined. The negative controls gave mutant frequencies within the range expected for the
L5178Y TK+/- mouse lymphoma cell line. All positive controls either for the induction of gene mutations or clastogenicity – methyl methanesulfonate (MMS), cyclophosphamide (CPP) and 7,12-dimethylbenz[a]anthracene (DMBA) - led to the expected increase in the frequencies of forward mutations. No cytotoxicity indicated by reduced relative total growth (RTG) of below 20% of control was observed in both experiments up to the highest applied concentration which showed clear test substance precipitation in culture medium.
Based on the results of the present study, the test substance did not cause any statistically significant or dose-dependent increase in the mutant frequencies either without S9 mix or after adding a metabolizing system in two experiments performed independently of each other.
Thus, under the experimental conditions described, the test item did not induce forward mutations or structural chromosome aberrations in vitro in the mouse lymphoma assay with L5178Y TK+/- cells in the absence and the presence of metabolic activation.
Referenceopen allclose all
Standard Plate Test
E.coli WP2 uvrA
| Dose [µg/plate] | Without S9 [Revertants/plate] |
With S9 [Revertants/plate] | ||
| Mean | Standard Deviation | Mean | Standard Deviation | |
| DMSO | 23.3 | 6.1 | 20.7 | 5.9 |
33 |
17.7 |
3.5 |
25.3 |
6.4 |
100 |
26.7 |
1.2 |
24.3 |
6.0 |
333 |
30.3 |
5.5 |
21.7 |
5.1 |
1000 |
17.7 |
4.2 |
16.7 |
2.5 |
2900 |
13.0 |
2.6 |
19.0 |
4.4 |
5800 |
6.0 |
2.0 |
5.3 |
1.5 |
5.0 (4 -NQO) |
1450.7 |
53.2 |
- |
- |
60 (2 -AA) |
- |
- |
109.7 |
12.9 |
TA 1535
Dose [µg/plate] |
Without S9 [Revertants/plate] |
With S9 [Revertants/plate] |
||
|
Mean |
Standard Deviation |
Mean |
Standard Deviation |
DMSO |
10.0 |
2.6 |
9.0 |
2.0 |
33 |
11.3 |
4.5 |
10.7 |
2.1 |
100 |
11.0 |
3.0 |
10.0 |
1.0 |
333 |
8.7 |
3.1 |
8.0 |
0.0 |
1000 |
9.3 |
3.2 |
6.3 |
3.2 |
2900 |
12.3 |
1.2 |
5.0 |
1.0 |
5800 |
7.0 |
1.0 |
4.0 |
1.0 |
5.0 (MNNG) |
3479.0 |
378.9 |
- |
- |
2.5 (2 -AA) |
- |
- |
232.0 |
9.8 |
TA 100
Dose [µg/plate] |
Without S9 [Revertants/plate] |
With S9 [Revertants/plate] |
||
|
Mean |
Standard Deviation |
Mean |
Standard Deviation |
DMSO |
91.3 |
19.0 |
82.3 |
0.6 |
33 |
93.3 |
5.9 |
85.7 |
10.1 |
100 |
109.3 |
15.6 |
96.0 |
1.0 |
333 |
114.7 |
21.5 |
100.7 |
8.4 |
1000 |
81.3 |
3.5 |
74.7 |
14.0 |
2900 |
69.7 |
6.7 |
84.3 |
7.2 |
5800 |
57.7 |
12.9 |
62.7 |
4.9 |
5.0 (MNNG) |
4008.7 |
386.0 |
- |
- |
2.5 (2 -AA) |
- |
- |
2567.7 |
19.9 |
TA 1537
Dose [µg/plate] |
Without S9 [Revertants/plate] |
With S9 [Revertants/plate] |
||
|
Mean |
Standard Deviation |
Mean |
Standard Deviation |
DMSO |
8.0 |
2.6 |
7.0 |
2.0 |
| 33 | 7.7 | 1.5 | 7.7 | 2.1 |
| 100 | 8.7 | 2.1 |
10.3 |
3.2 |
333 |
9.0 |
2.0 |
9.0 |
1.0 |
1000 |
6.3 |
1.2 |
5.0 |
0.0 |
2900 |
8.3 |
2.3 |
5.7 |
1.5 |
5800 |
2.3 |
0.6 |
2.3 |
0.6 |
100 (AAC) |
1219.3 |
211.6 |
- |
- |
2.5 (2 -AA) |
- |
- |
218.7 |
8.4 |
TA 98
Dose [µg/plate] |
Without S9 [Revertants/plate] |
With S9 [Revertants/plate] |
||
|
Mean |
Standard Deviation |
Mean |
Standard Deviation |
DMSO |
16.0 |
2.6 |
25.0 |
2.6 |
33 |
14.0 |
1.7 |
20.3 |
2.1 |
100 |
20.0 |
1.0 |
25.0 |
2.6 |
333 |
22.0 |
4.4 |
23.7 |
5.7 |
1000 |
15.0 |
3.6 |
21.0 |
1.7 |
2900 |
16.3 |
3.1 |
16.0 |
3.6 |
5800 |
12.0 |
2.0 |
7.7 |
1.5 |
10 (NOPD) |
539.3 |
32.7 |
- |
- |
2.5 (2 -AA) |
- |
- |
2113.3 |
90.0 |
Preincubation Test
TA 1535
Dose [µg/plate] |
Without S9 [Revertants/plate] |
With S9 [Revertants/plate] |
||
|
Mean |
Standard Deviation |
Mean |
Standard Deviation |
DMSO |
9.7 |
2.1 |
9.0 |
0.0 |
33 |
10.7 |
2.9 |
6.7 |
2.5 |
100 |
11.7 |
0.6 |
11.0 |
3.0 |
333 |
7.0 |
1.0 |
7.7 |
0.6 |
1000 |
9.7 |
1.5 |
7.3 |
2.3 |
2900 |
10.0 |
2.0 |
8.0 |
2.0 |
5800 |
2.3 |
1.5 |
3.7 |
0.6 |
5.0 (MNNG) |
5509.3 |
382.9 |
- |
- |
2.5 (2 -AA) |
- |
- |
332.0 |
16.5 |
TA 100
Dose [µg/plate] |
Without S9 [Revertants/plate] |
With S9 [Revertants/plate] |
||
|
Mean |
Standard Deviation |
Mean |
Standard Deviation |
DMSO |
104.0 |
13.9 |
110.0 |
5.6 |
33 |
113.7 |
9.1 |
105.3 |
6.7 |
100 |
109.0 |
12.3 |
116.0 |
6.6 |
333 |
111.7 |
10.0 |
105.0 |
12.3 |
1000 |
107.0 |
6.1 |
110.7 |
6.4 |
2900 |
89.0 |
20.2 |
90.3 |
6.4 |
5800 |
66.7 |
13.2 |
59.7 |
5.1 |
5.0 (MNNG) |
4232.7 |
364.5 |
- |
- |
2.5 (2 -AA) |
- |
- |
2966.0 |
49.9 |
TA 1537
Dose [µg/plate] |
Without S9 [Revertants/plate] |
With S9 [Revertants/plate] |
||
|
Mean |
Standard Deviation |
Mean |
Standard Deviation |
DMSO |
8.0 |
1.0 |
9.0 |
1.0 |
33 |
9.0 |
2.6 |
9.0 |
3.0 |
100 |
9.0 |
2.0 |
9.0 |
3.5 |
333 |
7.7 |
0.6 |
8.7 |
1.5 |
1000 |
8.3 |
2.3 |
6.3 |
0.6 |
2900 |
4.0 |
2.6 |
3.3 |
1.5 |
5800 |
1.7 |
0.6 |
2.0 |
1.0 |
100 (AAC) |
943.3 |
172.1 |
- |
- |
2.5 (2 -AA) |
- |
- |
257.7 |
18.5 |
TA 98
Dose [µg/plate] |
Without S9 [Revertants/plate] |
With S9 [Revertants/plate] |
||
|
Mean |
Standard Deviation |
Mean |
Standard Deviation |
DMSO |
27.0 |
10.4 |
28.3 |
4.0 |
33 |
22.0 |
4.4 |
36.0 |
2.0 |
100 |
27.3 |
7.4 |
33.7 |
1.2 |
333 |
26.3 |
3.8 |
33.3 |
6.0 |
1000 |
28.0 |
5.6 |
30.3 |
4.0 |
2900 |
19.3 |
4.5 |
25.3 |
5.1 |
5800 |
17.3 |
3.1 |
16.3 |
2.5 |
10 (NOPD) |
608.3 |
52.7 |
- |
- |
2.5 (2 -AA) |
- |
- |
2473.0 |
14.1 |
E.coli WP2 uvrA
Dose [µg/plate] |
Without S9 [Revertants/plate] |
With S9 [Revertants/plate] |
||
|
Mean |
Standard Deviation |
Mean |
Standard Deviation |
DMSO |
22.3 |
4.5 |
22.0 |
2.0 |
33 |
29.0 |
5.2 |
25.3 |
4.0 |
100 |
19.0 |
8.2 |
21.3 |
5.9 |
333 |
19.3 |
6.1 |
24.3 |
8.1 |
1000 |
20.7 |
1.5 |
22.0 |
4.4 |
2900 |
14.0 |
4.4 |
19.0 |
1.7 |
5800 |
10.0 |
2.0 |
4.7 |
2.9 |
5.0 (4 -NQO) |
746.3 |
69.6 |
- |
- |
60 (2 -AA) |
- |
- |
94.3 |
11.2 |
Summary of results - experimental results without metabolic activation
Exp. |
Exposure period (h) |
Test groups (µg/ml) | S9 mix | Precipitation | Cytotoxicty | Genotoxicity (colonies per 10^6 cells) | ||
| Relative cloning efficiency 1 RCE1 (%) | Relative total growth RTG (%) | Corrected mutant frequency (MFcorr.) | Mutant frequency threshold | |||||
| 1 | 4 | Vehicle control | - | n.d. | 100.0 | 100.0 | 48.2 | 174 |
| 6.25 | - | - | 92.5 | 84.2 | 61.1 | |||
12.50 |
- |
+ |
98.2 |
93.6 |
50.3 |
|
||
|
|
25.00 |
- |
+ |
84.6 |
84.2 |
58.6 |
|
|
|
50.00 |
- |
+ |
94.9 |
87.3 |
48.6 |
|
|
|
100.00 |
- |
+ |
98.2 |
84.7 |
49.5 |
|
|
|
200.00 |
- |
+ |
80.7 |
69.4 |
42.5 |
|
|
|
Positive control |
- |
n.d. |
75.9 |
35.5 |
734.8 |
|
2 |
24 |
Vehicle control |
- |
n.d. |
100.0 |
100.0 |
47.8 |
174 |
|
|
6.25 |
- |
- |
77.9 |
89.7 |
51.0 |
|
|
|
12.50 |
- |
- |
77.3 |
77.2 |
59.0 |
|
|
|
25.00 |
- |
- |
82.7 |
68.5 |
68.6 |
|
50.00 |
- |
- |
80.6 |
79.8 |
46.8 |
|
||
|
|
100.00 |
- |
+ |
76.6 |
66.2 |
52.5 |
|
|
|
200.00 |
- |
+ |
80.6 |
48.9 |
60.1 |
|
|
|
Positive control |
- |
n.d. |
73.0 |
57.0 |
522.6 |
|
Summary of results - experimental results with metabolic activation
|
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Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Bacterial Mutagenicity:
The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay (TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA) in a dose range of 33 μg - 5800 μg/plate (SPT) and 33 μg - 5800 μg/plate (PIT). The Standard plate test (SPT) and preincubation test (PIT) were performed both with and without metabolic activation (liver S9 mix from induced rats). Precipitation of the test substance was found from about 333 μg/plate onward in the standard plate test and from about 1000 μg/plate onward in the preincubation test with and without S9 mix. A bacteriotoxic effect was observed depending on the strain and test conditions from about 2900 μg/plate onward. A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.
Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.
Mammalian Cytogenicity:
The test item, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments. In each experimental group two parallel cultures were analyzed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage. The highest applied concentration in this study (2384 μg/mL of the test item) was chosen with regard to the purity (83.9%) and the solubility properties of the test item and with respect to the current OECD Guideline 487. Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 487. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentrations, which showed precipitation. In the absence and presence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to precipitating concentrations.
Mammalian Mutagenicity:
The test substance was tested for its ability to induce gene mutations at the thymidine kinase (TK) locus or structural chromosome aberrations at chromosome 11 in L5178Y TK+/- mouse lymphoma cells in vitro with the microwell method. Two independent experiments were carried out with and without the addition of liver S9 mix from phenobarbital and -naphthoflavone induced rats (exogenous metabolic activation).
According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following concentrations were tested and evaluated for gene mutations.
1st Experiment
without S9 mix (4-hour exposure period)
0; 6.25; 12.50; 25.00; 50.00; 100.00; 200.00 μg/mL
with S9 mix (4-hour exposure period)
0; 6.25; 12.50; 25.00; 50.00; 100.00; 200.00 μg/mL
2nd Experiment
without S9 mix (24-hour exposure period)
0; 6.25; 12.50; 25.00; 50.00; 100.00; 200.00 μg/mL
with S9 mix (4-hour exposure period)
0; 4.69; 9.38; 18.75; 37.50; 75.00; 150.00 μg/mL
Cells were treated with the test substance for 4 and 24 hours in the absence of metabolic activation and for 4 hours in the presence of metabolic activation. Subsequently, cells were cultured for an expression period of about 48 hours and then cultured in selection medium for another approx. 10 days. Finally, the number of large and small colonies was determined. The negative controls gave mutant frequencies within the range expected for the
L5178Y TK+/- mouse lymphoma cell line. All positive controls either for the induction of gene mutations or clastogenicity – methyl methanesulfonate (MMS), cyclophosphamide (CPP) and 7,12-dimethylbenz[a]anthracene (DMBA) - led to the expected increase in the frequencies of forward mutations. No cytotoxicity indicated by reduced relative total growth (RTG) of below 20% of control was observed in both experiments up to the highest applied concentration which showed clear test substance precipitation in culture medium.
Based on the results of the present study, the test substance did not cause any statistically significant or dose-dependent increase in the mutant frequencies either without S9 mix or after adding a metabolizing system in two experiments performed independently of each other.
Thus, under the experimental conditions described, the test item did not induce forward mutations or structural chromosome aberrations in vitro in the mouse lymphoma assay with L5178Y TK+/- cells in the absence and the presence of metabolic activation.
Justification for classification or non-classification
As there is no indication for genotoxic potential, classification for genotoxicity is not warranted according to the EC Directive (No.93/21/EEC) and CLP (No. 1272/2008 of 16 December 2008).
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
