Registration Dossier
Registration Dossier
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EC number: 947-918-6 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
In vitro gene mutation in bacteria (OECD 471): negative in S. thyphimurium TA 98, TA 102, TA 100, TA 1535 ans TA 1537 with and without metabolic activation
in vitro mammalian cell micronucleus test (OECD 487): negative in Chinese hamster lung fibroblasts (V79) with and without metabolic activation
In vitro gene mutation in mammalian cells (OECD 490): negative in L5178Y cells 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:
- 20 Feb - 04 Apr 2018
- 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:
- adopted in 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- adopted in 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- GLP-Landesleitstelle Bayern, Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany
- 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
- Remarks:
- not applicable
- Details on mammalian cell type (if applicable):
- not applicable
- Additional strain / cell type characteristics:
- DNA polymerase A deficient
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats, induced with phenobarbital and beta-naphthoflavone
- Test concentrations with justification for top dose:
- The test item was tested in the pre-experiment with tester strains TA 98 and TA 100 with the following concentrations:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
No cytotoxicity was observed.
Experiment I - Plate Incorporation Method (all tester strains): 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
The maximum concentration was chosen as recommended in the guideline followed.
Experiment II - Plate Incorporation Method (all tester strains): 15.8, 50.0, 158, 500, 1580 and 5000 µg/plate
The maximum concentration was chosen as recommended in the guideline followed. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Tetrahydrofuran (THF)
- Lot/Batch: 17A261139
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity. - Untreated negative controls:
- yes
- Remarks:
- A. dest.
- Negative solvent / vehicle controls:
- yes
- Remarks:
- THF
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- methylmethanesulfonate
- other: 4-nitro-o-phenylene-diamine (4-NOPD): -S9: 10 µg/plate (in DMSO) for TA 98 and 40 µg/plate (in DMSO) for TA 1537; 2-aminoanthracene (2-AA): +S9: 2.5 µg/plate (in DMSO) for TA 98, TA 100, TA 1535, TA 1537 and 10 µg/plate (in DMSO) for TA 102
- Remarks:
- Biological activity of the S9 mix in the Salmonella typhimurium assay was assessed using 2-aminoanthracene and benzo[a]pyrene.
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation for experiments I and II)
DURATION
- Exposure duration: at least 48 h (at 37 °C)
NUMBER OF REPLICATIONS:
triplicate in two independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: Inspection of clearing or diminution of the background lawn or of reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control - Evaluation criteria:
- VALIDITY CRITERIA:
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100, TA 102)
- the negative control plates (A. dest.) +/-S9 mix are within the historical control data range
- corresponding background growth on negative control, solvent control and test plates is observed
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable.
EVALUATION CRITERIA:
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
A test item producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system. - Statistics:
- The colonies were counted using a ProtoCOL counter (Meintrup DWS Laborgeräte GmbH). If precipitation of the test item precluded automatic counting the revertant colonies were counted by hand. In addition, tester strains with a low spontaneous mutation frequency like TA 1535 and TA 1537 were counted manually. Mean values and standard deviations of each treatment and control (negative, positive and solvent) were calculated.
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- 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: Results of positive and negative controls fell within historical control data range. Data are summarised in "Any other information on results incl. tables". - Conclusions:
- Under the conditions of the Ames Assay the substance was not mutagenic in any of the five bacterial strains (TA1535, TA1537, TA98, TA100 and TA102) with and without metabolic activation tested.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 08 March - 27 June 2018
- 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 29 July 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany
- Type of assay:
- in vitro mammalian cell micronucleus test
- Target gene:
- Not applicable
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Eurofins Munich stock cultures
- Suitability of cells: Cells were chosen because of their stable karyotype and their low spontaneous induction rate of micronucleus formation under standardised culture conditions
MEDIA USED
- Minimum essential medium (MEM) supplemented with:
10% fetal bovine serum (FBS)
100 U/100 μg/mL penicillin/streptomycin solution
2 mM L-glutamine
2.5 μg/mL amphotericin
25 mM HEPES
Treatment Medium (short-term exposure): MEM medium with 0% FBS
After Treatment Medium / Treatment Medium (long-term exposure): MEM medium with 10% FBS and 1.5 μg/mL cytochalasin B
- Properly maintained: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Wistar rats treated with phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw)
- Test concentrations with justification for top dose:
- Pre-experiment: 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 2000 and 2500 μg/mL
Experiment I (short-term exposure 4 h, without and with metabolic activation): 0.5, 1.0, 2.5, 5, 10, 15 and 25 μg/mL
Experiment II (long-term exposure 24 h, without metabolic activation): 1.0, 2.5, 5, 10, 15, 25, 50 and 100 μg/mL
The selection of the concentrations was based on data from the pre-experiment. Precipitation of the test item was noted at 15 μg/mL and higher with and without metabolic activation in experiment I and at 25 μg/mL and higher in experiment II in the cultures at the end of treatment. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: THF 0.5% v/v in cell culture medium
- Justification for choice of solvent/vehicle: It was not possible to prepare a solution of the test item with cell culture medium. Therefore, the test item was dissolved in tetrahydrofuran (THF) and diluted in cell culture medium to reach a final concentration of 0.5% v/v THF and the final test item concentrations in the samples. The solvent was compatible with the survival of the cells and the S9 activity. - Untreated negative controls:
- yes
- Remarks:
- cell culture medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- cell culture medium with 0.5% (v/v) THF
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- methylmethanesulfonate
- other: colchicine
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding: 5 x 10E05 cells
DURATION
- Preincubation period: approx. 48 h
- Exposure duration: 4 h (experiment I), 24 h (experiment II)
- Cytochalasin B exposure: 20 h (experiment I), 23 h (experiment II):
- Preparation interval: 24 h (experiment I and II):
- Total culture period (exposure started 48 h after culture initiation): 72 h (experiment I and II)
NUMBER OF REPLICATIONS:
Duplicate cultures were performed at each concentration level
CONCENTRATIONS FOR MICROSCOPIC ANALYSES:
The following concentrations were selected for the microscopic analyses of micronuclei frequencies. The selection of the maximum concentration was based on occurrence of precipitation of the test item for all experimental conditions.
Experiment I (short-term exposure 4 h, without and with metabolic activation): 5, 10 and 15 μg/mL
Experiment II (long-term exposure 24 h, without metabolic activation): 10, 15 and 25 μg/mL
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
After cultivation, the complete culture medium was removed and cells were trypsinated and resuspended in about 9 mL complete culture medium. The cultures were transferred into tubes and incubated with hypotonic solution (0.4% KCl) for some minutes at room temperature. After the treatment with the hypotonic solution the cells were fixed with methanol and glacial acetic acid (3 + 1). The cells were resuspended gently and the suspension was dropped onto clean glass slides. The cells were then dried on a heating plate. Finally, the cells were stained with acridine orange solution.
NUMBER OF CELLS EVALUATED:
At least 2000 binucleated cells per concentration (1000 binucleated cells per slide) were analysed.
CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
Clearly surrounded by a nuclear membrane, having an area of less than one-third of that of the main nucleus, being located within the cytoplasm of the cell and not linked to the main nucleus via nucleoplasmic bridges. Mononucleated and multinucleated cells and cells with more than six micronuclei were not considered.
DETERMINATION OF CYTOTOXICITY
- Method: cytokinesis block proliferation index (CBPI)
- Any supplementary information relevant to cytotoxicity: refer to section 'Any other information on materials and methods incl. tables' - Evaluation criteria:
- A test item is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
- the increase is concentration-related in at least one experimental condition when evaluated with an appropriate trend test
- any of the results are outside the distribution of the historical negative/solvent control data (e.g. Poisson-based 95% control limits).
When all of these criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system. A test item is considered to be clearly negative if in all experimental conditions examined none of the criteria mentioned above are met. - Statistics:
- The nonparametric χ² Test was performed to verify the results in both experiments. The χ² Test for trend was performed to test whether there is a concentration-related increase in the micronucleated cells frequency in the experimental conditions.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES:
The following concentrations were tested with and without S9 mix: 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 2000 and 2500 μg/mL.
The concentration of 2500 μg/mL was considered to be the highest test concentration used in this test system following the recommendation of the corresponding OECD testing guideline 487 and based on the physico-chemical properties of the test item. Since the organic solvent THF was used which can only be applied at a final concentration of 0.5% (v/v) in cell culture, the maximum technically feasible concentration used in this study was determined to be 2500 μg/mL.
HISTORICAL CONTROL DATA:
Refer to section 'Any other information on results incl. tables' and to attached background information.
ADDITIONAL INFORMATION:
Refer to section 'Any other information on results incl. tables' and to attached background information. - Conclusions:
- The test item is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Test.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 13 Mar - 04 Apr 2018
- 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 in 2016
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- adopted in 2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany
- Type of assay:
- other: in vitro mammalian cell gene mutation tests using the thymidine kinase gene (migrated information)
- Target gene:
- Thymidine kinase locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Eurofins Munich stock cultures
- Suitability of cells: Cell line L5178Y is recommended in the OECD guideline 490.
- Cell cycle length, doubling time or proliferation index: 10-12 h doubling time; cloning efficiency of > 50%
- Methods for maintenance in cell culture if applicable: Stock cultures of cleansed L5178Y cell line are stored over liquid nitrogen (vapour phase). Thawed stock cultures are maintained in plastic culture flasks in RPMI 1640 complete medium and subcultured three times per week.
- Modal number of chromosomes: 40 ± 2 chromosomes
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 complete medium supplemented with 5% horse serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes - Additional strain / cell type characteristics:
- not applicable
- 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:
- Preliminary toxicity test: 2.5, 5.0, 10, 50, 150, 500, 1000 and 2000 μg/mL with and without metabolic activation
Main experiment: 2.5, 5.0, 10, 50, 150, 250 and 500 μg/mL with and without metabolic activation
Due to the occurrence of precipitation at 500 μg/mL (without and with metabolic activation), thus this concentration was selected as the highest. - Vehicle / solvent:
- - Vehicle/solvent used: tetrahydrofurane (THF)
- Justification for choice of solvent/vehicle: Based on an pre-experiment for solubility and due to the nature of the test item, it was not possible to prepare a solution of the test item with an appropriate solvent and with the recommended concentration of 5 mg/mL. By lowering the highest concentration to 2 mg/mL it was possible to prepare a solution, and the best suited vehicle was THF. - Untreated negative controls:
- yes
- Remarks:
- treatment medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- tertrahydrofurane (0.25% v/v)
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- ethylmethanesulphonate
- methylmethanesulfonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 12 days
NUMBER OF REPLICATIONS: single cultures in one experiment
SELECTION AGENT (mutation assays): trifluorothymidine (TFT)
DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (RSG), relative cloning efficiency (RCE) and relative total growth (RTG)
OTHER:
Small and large colonies were differentiated, as small colonies are capable to indicate chromosomal mutations. - Evaluation criteria:
- The test item is considered mutagenic if the following criteria are met:
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 10E6 cells and
- A dose-dependent increase in mutant frequency is detected.
Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation of the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend of the test is negative. - Statistics:
- The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls. Mutant frequencies of the solvent/negative controls were used as reference.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: The pH-value detected with the test item and the osmolality were within the physiological range.
- Precipitation: Precipitation of the test item was noted in the pre- and the main experiment at concentrations of 500 μg/mL and higher.
RANGE-FINDING/SCREENING STUDIES: The toxicity of the test item was determined in a pre-experiment up to a maximum concentration of 2 mg/mL. Eight concentrations (2.5, 5.0, 10, 50, 150, 500, 1000 and 2000 μg/mL) were tested without and with metabolic activation under same conditions as for the main experiment. No growth inhibition was observed with and without metabolic activation. The selection of the concentrations used in the main experiment was based on data from the pre-experiment. Due to the occurrence of precipitation at 500 μg/mL (without and with metabolic activation), this was selected as the highest concentration.
HISTORICAL CONTROL DATA: see Table 2 in 'Any other information on results incl. tables'
ADDITIONAL INFORMATION ON CYTOTOXICITY:
No growth inhibition was observed in the experiment without and with metabolic activation. - Conclusions:
- Under the experimental conditions of the gene mutation assay the test item did not induce gene mutations at the TK locus in L5178Y cells with and without metabolic activation.
Referenceopen allclose all
Due to the amount and complexity of the results tables, all additional data are attached in a pdf document (Tabulated results.pdf). Only the historical laboratory control data are summarised below.
Table 1: Historical Laboratory Control Data of the Negative Control (in 2014 - 2016) without S9 (-S9)
|
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
TA 102 |
Mean |
24.2 |
90.7 |
13.8 |
8.2 |
270.4 |
SD |
6.7 |
15.6 |
6.7 |
2.9 |
55.0 |
Min |
11 |
49 |
4 |
3 |
141 |
Max |
58 |
155 |
41 |
35 |
472 |
RSD [%] |
27.7 |
17.2 |
48.6 |
35.3 |
20.3 |
n |
972 |
1191 |
929 |
931 |
682 |
Table 2: Historical Laboratory Control Data of the Positive Control (in 2014
- 2016) without S9 (-S9)
|
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
TA 102 |
Substance Conc./plate |
4-NOPD 10 µg |
NaN3 10 µg |
NaN3 10 µg |
4-NOPD 40 µg |
MMS 1 µL |
Mean |
430.7 |
612.1 |
792.0 |
94.5 |
1729.2 |
SD |
155.5 |
220.0 |
299.5 |
22.7 |
518.8 |
Min |
141 |
132 |
38 |
35 |
272 |
Max |
1830 |
1423 |
1854 |
273 |
3321 |
RSD [%] |
36.1 |
35.9 |
37.8 |
24.0 |
30.0 |
n |
971 |
1188 |
931 |
929 |
682 |
Table 3: Historical Laboratory Control Data of the Negative Control (in 2014 - 2016) with S9 (+S9)
|
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
TA 102 |
Mean |
29.0 |
96.4 |
10.5 |
8.3 |
339.7 |
SD |
6.8 |
14.1 |
4.5 |
3.1 |
71.3 |
Min |
15 |
62 |
3 |
3 |
157 |
Max |
59 |
160 |
38 |
36 |
586 |
RSD [%] |
23.4 |
14.6 |
42.7 |
37.4 |
21.0 |
n |
967 |
1189 |
925 |
926 |
676 |
Table 4: Historical Laboratory Control Data of the Positive Control (in 2014
- 2016) with S9 (+S9)
|
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
TA 102 |
Substance Conc./plate |
2-AA 2.5 µg |
2-AA 2.5 µg |
2-AA 2.5 µg |
2-AA 2.5 µg |
2-AA 10 µg |
Mean |
1880.5 |
1727.7 |
133.9 |
234.1 |
801.2 |
SD |
708.5 |
522.0 |
134.9 |
101.4 |
223.7 |
Min |
70 |
169 |
22 |
26 |
137 |
Max |
3606 |
3132 |
1954 |
682 |
3588 |
RSD [%] |
37.7 |
30.2 |
100.8 |
43.3 |
27.9 |
n |
966 |
1184 |
927 |
925 |
678 |
S9: metabolic activation
Mean: mean of revertants/plate
Min.: minimum of revertants/plate
Max.: maximum of revertants/plate
SD: Standard Deviation
RSD: Relative Standard Deviation
n: Number of control values
Table 1: Summary: Experiment I and II, without metabolic activation
|
Dose group |
Concentration [µg/mL] |
No. of cells evaluated |
Cytostasis [%] |
Relative cell growth [%] |
Micronucleated ceslls frequency [%] |
Historical control limits negative control |
P |
Statistically significant increasea |
Experiment I 4 h treatment, 24 h fixation interval |
C |
0 |
2000 |
0* |
101 |
0.55 |
0.37% - 1.37% |
/ |
/ |
S |
0 |
4000 |
0 |
100 |
0.53 |
/ |
/ |
||
4 |
5 |
4000 |
0* |
102 |
0.53 |
- |
- |
||
5 |
10 |
4000 |
3 |
97 |
0.83 |
- |
- |
||
6 |
15 |
4000 |
5 |
95 |
0.68 |
+ |
- |
||
MMS |
25 |
2000 |
18 |
82 |
3.40 |
- |
+ |
||
Colc |
2.0 |
2000 |
0 |
109 |
2.90 |
- |
+ |
||
|
|||||||||
Experiment II 24 h treatment, 24 h fixation interval |
C |
0 |
2000 |
0 |
154 |
1.05 |
0.37% - 1.37% |
/ |
/ |
S |
0 |
2000 |
0 |
100 |
1.30 |
/ |
/ |
||
4 |
10 |
2000 |
7 |
93 |
0.70 |
- |
- |
||
5 |
15 |
2000 |
14 |
86 |
0.95 |
- |
- |
||
6 |
25 |
2000 |
28 |
72 |
0.80 |
+ |
- |
||
MMS |
25 |
2000 |
0 |
120 |
7.50 |
- |
+ |
||
Colc |
0.16 |
2000 |
33 |
67 |
8.00 |
- |
+ |
||
Table 2: Summary: Experiment I, with metabolic activation
|
Dose group |
Concentration [µg/mL] |
No. of cells evaluated |
Cytostasis [%] |
Relative cell growth [%] |
Micronucleated ceslls frequency [%] |
Historical control limits negative control |
P |
Statistically significant increasea |
Experiment I 4 h treatment, 24 h fixation interval |
C |
0 |
4000 |
0* |
112 |
0.90 |
0.42% - 1.64% |
/ |
/ |
S |
0 |
2000 |
0 |
100 |
0.70 |
/ |
/ |
||
4 |
5 |
4000 |
0* |
103 |
0.53 |
- |
- |
||
5 |
10 |
2000 |
0* |
102 |
0.85 |
- |
- |
||
6 |
15 |
2000 |
2 |
98 |
0.75 |
+ |
- |
||
CPA |
2.5 |
2000 |
36 |
64 |
4.50 |
- |
+ |
C: Negative Control (Culture medium)
S: Solvent Control (THF 0.5% v/v in culture medium)
a: statistical significant increase compared to solvent control (χ² test , p<0.05)
+: significant
-: not significant
MMS: Methylmethanesulfonate, Positive Control (without metabolic activation) [25 μg/mL]
Colc: Colchicine, Positive Control (without metabolic activation) [0.16 and 2.0 μg/mL]
CPA: Cyclophosphamide, Positive Control (with metabolic activation) [2.5 μg/mL]
CBPI: Cytokinesis Block Proliferation Index, CBPI = ((c1 x 1) + (c2 x 2) + (cx x 3))/n
Relative Cell Growth: 100 x ((CBPI Test conc – 1) / (CBPI control -1))
c1: mononucleate cells
c2: binucleate cells
cx: multinucleate cells
n: total number of cells
P: Precipitation
Cytostasis [%] = 100- Relative Cell Growth [%]
*: the cytostasis is defined 0, when the relative cell growth exceeds 100%
Table 3: Historical Laboratory Control Data of the negative, solvent and positive control in Chinese hamster V79 cells (in 2012-2017)
|
Negative control |
||||
Metabolic activation |
|||||
Without |
with |
||||
Mean |
0.87 |
1.03 |
|||
SD |
0.25 |
0.31 |
|||
RSD |
28.75 |
29.76 |
|||
Min |
0.45 |
0.55 |
|||
Max |
1.50 |
1.75 |
|||
LCL |
0.37 |
0.42 |
|||
UCL |
1.37 |
1.64 |
|||
n |
62 |
35 |
|||
|
|||||
|
Solvent control |
||||
Metabolic activation |
|||||
Without |
with |
||||
Mean |
0.97 |
1.05 |
|||
SD |
0.25 |
0.35 |
|||
RSD |
25.87 |
33.49 |
|||
Min |
0.55 |
0.55 |
|||
Max |
1.40 |
1.8 |
|||
LCL |
0.47 |
0.35 |
|||
UCL |
1.48 |
1.75 |
|||
n |
33 |
17 |
|||
|
Positive control |
||||
Metabolic activation |
|||||
Without |
With |
||||
MMS |
Colchicine |
CPA |
|||
Mean |
4.43 |
4.18 |
3.99 |
||
SD |
1.86 |
2.27 |
1.14 |
||
RSD |
41.93 |
54.30 |
28.54 |
||
Min |
2.10 |
1.85 |
2.25 |
||
Max |
8.65 |
12.30 |
6.25 |
||
LCL |
0.72 |
0.00 |
1.71 |
||
UCL |
8.15 |
8.72 |
6.27 |
||
n |
18 |
62 |
35 |
||
Negative Control: Cell culture medium
Solvent Control: DMSO 1% v/v or ethanol 0.5% v/v in cell culture medium
MMS: Positive Control-clastogenicity without metabolic activation: Methylmethanesulfonate
Colchicine: Positive Control- aneugenicity without metabolic activation
CPA: Positive Control-clastogenicity with metabolic activation: Cyclophosphamide
Mean: Mean number of micronucleated cells (%)
SD: Standard Deviation
RSD: Relative Standard Deviation (%)
Min: Minimum number of micronucleated cells (%)
Max: Maximum number of micronucleated cells (%)
LCL: Lower control limit (95%, mean-2SD)
UCL: Upper control limit (95%, mean+2SD)
n: Number of assays
Table 1. Summary of results of main experiment with and without metabolic activation
| Test Group | Conc. [µg/mL] | RCEa[%] | RTGb[%] | MFc[mutants/ 10E6 cells] | IMFd[mutants/ 10E6 cells] | GEFe exceeded | Statistical significant increasef | Precipitate | |
| Exp without S9 | C1 | 0 | 94.9 | 101.7 | 87.3 | / | / | - | - |
| C2 | 115.9 | 122.2 | / | / | - | ||||
| S1 | 0 | 100 | 100 | 99.4 | / | / | / | - | |
| S2 | / | / | / | - | |||||
| 1 | 2.5 | 97.9 | 105.5 | 82 | -17.4 | - | - | - | |
| 2 | 5 | 90.6 | 95.4 | 77.1 | -22.3 | - | - | - | |
| 3 | 10 | 79.2 | 79.8 | 111.3 | 11.9 | - | - | - | |
| 4 | 50 | 80.3 | 84.9 | 129.4 | 30 | - | - | - | |
| 5 | 150 | 92 | 95.3 | 107.9 | 8.5 | - | - | - | |
| 6 | 250 | 108 | 112.8 | 58.1 | -41.3 | - | - | - | |
| 7 | 500 | 90.6 | 98.9 | 150 | 50.6 | - | + | + | |
| EMS | 300 | 58.7 | 61.2 | 1180.1 | 1080.7 | + | + | - | |
| MMS | 10 | 53.9 | 46.3 | 1124.8 | 1025.4 | + | + | - | |
| Exp with S9 | C1 | 0 | 100.2 | 88.9 | 105.5 | / | / | - | - |
| C2 | 92.9 | 93.1 | / | / | - | ||||
| S1 | 0 | 100 | 100 | 103.4 | / | / | / | - | |
| S2 | / | / | / | - | |||||
| 1 | 2.5 | 95.8 | 96.8 | 95.4 | -8 | - | - | - | |
| 2 | 5 | 117.8 | 124.9 | 112.9 | 9.5 | - | - | - | |
| 3 | 10 | 108.5 | 109.8 | 117.9 | 14.5 | - | - | - | |
| 4 | 50 | 131 | 143 | 88.2 | -15.2 | - | - | - | |
| 5 | 150 | 80.3 | 79.3 | 134.9 | 31.5 | - | - | - | |
| 6 | 250 | 105.1 | 110.6 | 82.2 | -21.2 | - | - | - | |
| 7 | 500 | 106.7 | 110.1 | 85.7 | -17.7 | - | - | + | |
| B[a]P | 1.5 | 82.6 | 35 | 463.7 | 360.3 | + | + | - | |
C: Negative Controls
S: Solvent Controls (THF 0.25%)
a: Relative Cloning Efficiency, RCE = [(CEdose group/ CEof corresponding controls)x100]Cloning Efficiency, CE = ((-LN (((96 - (mean P1,P2)) / 96)) / 1.6) x100)
b: Relative Total Growth, RTG = (RSGxRCE)/100
c: Mutant Frequency, MF = {-ln [negative cultures/total wells (selective medium)] / -ln [negative cultures/total wells (non selective medium)]}x800
d: Induced Mutant Frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls
e: Global Evaluation Factor, GEF (126); +: GEF exceeded, -: GEF notexceeded
f: Statistical significant increase in mutant frequency compared to solvent controls (Mann Whitney test, p<0.05). +: significant; -not significant
EMS: Ethylmethanesulfonate
MMS: Methylmethanesulfonate
B[a]P: Benzo[a]pyrene
Table 2. Historical control data from Jan 2011 to Dec 2016
| NC | PC | THF | |||||
| -S9 | +S9 | EMS | MMS | B[a]P | -S9 | +S9 | |
| (300 µg/mL) | (10 µg/mL) | (1.5 µg/mL) | |||||
| Mean | 87.9 | 85.1 | 726.5 | 763.4 | 535.5 | 105.9 | 108.6 |
| Min | 50.1 | 50.1 | 318.7 | 376.4 | 312.4 | 56.9 | 69.2 |
| Max | 170.3 | 165.9 | 2919 | 2416.1 | 1108.9 | 153 | 168.8 |
| SD | 25.5 | 24.3 | 203.5 | 421.6 | 152.5 | 30 | 23.5 |
| RSD [%] | 29 | 28.6 | 28 | 55.2 | 28.5 | 28.4 | 21.7 |
| n = | 447 | 653 | 211 | 254 | 57 | 18 | 18 |
NC: negative control
PC: positive controls (+S9: B[a]P, -S9: EMS, MMS)
S9: metabolic activation
Mean: mean of mutant frequency [mutants/ 10E6 cells]
Min.: minimum of mutant frequency [mutants/10E6 cells]
Max.: maximum of mutant frequency [mutants/10E6 cells]
SD: standard deviation
RSD: relative standard deviation
n: number of control values
EMS: Ethylmethanesulfonate
MMS: Methylmethanesulfonate
B[a]P: Benzo[a]pyrene
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Mutagenicity in bacteria in vitro
A bacterial gene mutation assay (Ames test) was performed with Reaction product of 1,3,5-Triazine-2,4,6-triamine, polymer with formaldehyde, methylated and C16-18 fatty alcohols according to OECD guideline 471 and in compliance with GLP (Schreib, 2018). S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 were tested using the plate incorporation method in two independent experiments. Experiments were performed in the absence and presence of metabolic activation (phenobarbital and beta-naphthoflavone induced rat liver S9-mix) in 3 replicates each, up to the limit concentration of 5000 µg/plate. No cytotoxicity and no precipitation were observed at any concentration in any experiment. Therefore, the maximum concentration of 5000 µg/plate was chosen as recommended in the guideline followed. 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, the test substance 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.
Cytogenicity / micronucleus study in vitro
The potential of Reaction product of 1,3,5-Triazine-2,4,6-triamine, polymer with formaldehyde, methylated and C16-18 fatty alcohols to induce the formation of micronuclei in mammalian cells was investigated in a study according to OECD guideline 487 under GLP conditions (Donath, 2018). The selection of the concentrations used in experiment I and II was based on data from a pre-experiment. In experiment I 15 μg/mL without and with metabolic activation and in experiment II 25 μg/mL were selected as highest concentration for the microscopic analysis of micronuclei. The cells were prepared 24 h after start of treatment with the test item. The treatment intervals were 4 h without and with metabolic activation (experiment I) and 24 h without metabolic activation (experiment II). Duplicate cultures were set up and 1000 binucleated cells per culture were scored for micronuclei. The following concentrations were evaluated for micronuclei frequencies: experiment I (short-term exposure 4 h, without and with metabolic activation): 5, 10 and 15 μg/mL and experiment II (long-term exposure 24 h, without metabolic activation): 10, 15 and 25 μg/mL. The test item was dissolved in THF and rediluted in cell culture medium at a ratio of 1:200 to achieve the final test item concentrations and a final THF concentration of 0.5% (v/v). Precipitation of the test item was noted at 15 μg/mL and higher with and without metabolic activation in experiment I and at 25 μg/mL and higher in experiment II in the cultures at the end of treatment. In experiment I and II with and without metabolic activation no increase of the cytostasis above 30% was noted. No statistically significant increase of cells with micronuclei was noted in the concentration groups of the test item evaluated in experiment I and II with and without metabolic activation. Methylmethanesulfonate (MMS, 25 μg/mL) and cyclophosphamide (CPA, 2.5 μg/mL) were used as clastogenic controls. Colchicine (0.16 and 2.0 μg/mL) was used as aneugenic control. All positive control substances induced distinct and statistically significant increases of the micronucleus frequency, thus demonstrates the validity of the assay. In conclusion, the test item is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Test.
Mutagenicity in mammalian cells in vitro
An in vitro mammalian cell gene mutation assay was performed with Reaction product of 1,3,5-Triazine-2,4,6-triamine, polymer with formaldehyde, methylated and C16-18 fatty alcohols to assess its potential to induce gene mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The study was conducted according to OECD guideline 490 under GLP conditions (Voges, 2018). Based on data from a pre-experiment, concentrations of 2.5, 5.0, 10, 50, 150, 250 and 500 μg/mL were tested with and without metabolic activation. Precipitation of the test item was noted in the pre- and the main experiment at the concentration of 500 μg/mL. No cytotoxicity and no relevant increases of induced mutant frequencies were observed with and without metabolic activation. Appropriate positive controls (EMS, MMS and B[a]P) significantly increased the number of small colonies, thus providing the efficiency of the test system. In conclusion, under the experimental conditions reported, the test item is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.
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) No. 1272/2008, and are, therefore, conclusive but not sufficient for classification.
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