Registration Dossier
Registration Dossier
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EC number: 244-479-6 | CAS number: 21615-47-4
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Ames test (OECD 471): negative with S. typhimurium TA 98, TA 100, TA 1535 and TA 1537 and E.coli WP2 uvr A with and without metabolic activation
Chomosome aberration test (OECD 473): negative in CHO/IU cells with and without metabolic activation
HPRT (OECD 476): negative in CHO cells with and wihout 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:
- 03 -13 Mar 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:
- adopted in 1997
- Deviations:
- yes
- Remarks:
- missing DMSO (solvent for positive controls) control
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon (for S. typhimurium strains)
trp operon (for E. coli strain) - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital (one time at 30 mg/kg bw and three times at 60 mg/kg bw) and 5,6-benzoflavone (one time at 80 mg/kg bw)
- Test concentrations with justification for top dose:
- The test substance was corrected by the purity (50%) before preparation of dosing solutions.
Experiment 1: 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/mL
Experiment 2: 313, 625, 1250, 2500 and 5000 µg/mL - Vehicle / solvent:
- - Vehicle/solvent used: destilled water (test substance and NaN3), DMSO (AF-2, ICR-191 and 2AA)
- Justification for choice of solvent/vehicle: The solvent was chosen based on a good solubililty and stability (no change in color, exothermic reaction nor gas generation at room temperature within 4 h) at 50 mg/mL . - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- other: 2-Aminoanthracene (2AA); 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamine (AF-2); ICR-191
- Remarks:
- Efficasy of S9-mix was characterised with benzo(a)pyrene and 9,10-dimethylanthracene (mutagens that requires metabolic activation by microsomal enzymes).
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: preincubation
- Cell density at seeding:
Experiment 1: 2.9, 3.2, 6.9, 4.5 and 3.7 x10E9 cells/mL for TA100, TA1535, WP2uvrA, TA98 and TA1535, respectively
Experiment 2: 2.5, 3.2, 6.6, 4.7 and 3.4 x10E9 cells/mL for TA100, TA1535, WP2uvrA, TA98 and TA1535, respectively
DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: Triplicates each in 2 independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn observed using a stereomicroscope
- OTHER: All plates were counted with a colony analyzer (CA-1 lD, SYSTEM SCIENCE). Square correction and miscounting correction were conducted when counting with the colony analyzer. - Evaluation criteria:
- The test substance was judged to be positive when the number of revertant colonies increased to twice or more than that in the negative control and when the responses were dose-related and/or reproducible.
- Statistics:
- Mean values and standard deviations were calculated.
- 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:
- not applicable
- 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:
- not applicable
- 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:
- not applicable
- 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:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- 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:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test substance was observed in either the presence or absence of S9 mix.
HISTORICAL CONTROL DATA : The test results showed that the numbers of revertant colonies in the negative and positive controls were within the range of the historical data at the testing facility.
ADDITIONAL INFORMATION ON CYTOTOXICITY: Bacterial growth inhibition was not observed at any test condition. - Conclusions:
- Under the conditions of the conducted test the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA) tested with and without metabolic activation.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 13 Mar - 19 Jun 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 29 Jul 2016
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- other: in vitro mammalian chromosome aberration test
- Target gene:
- not applicable
- Species / strain / cell type:
- other: Chinese hamster lung fibroblasts (CHL/IU cells)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Health Science Research Resources Bank, Japan Health Sciences Foundation
- Doubling time: 15 h
- Number of passages: 7 (cell growth inhibition test) and 19 (chromosomal aberration test)
- Methods for maintenance in cell culture if applicable: 37°C, humid conditions, 5% CO2, 2-3 passages per week
- Modal number of chromosomes: 25 per cell
MEDIA USED
- Type and identity of media: Eagle's minimum essential medium supplemented with L-Glutamine (final concentration: 0.292 g/L), sodium hydrogen carbonate (final concentration: approx. 1.95 g/L) and 10 vol% heat-inactivated NBCS
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes, negative - Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats treated with phenobarbital/5,6-benzoflavone
- Test concentrations with justification for top dose:
- Cell growth inhibition test
Short-term treatment and 24-h treatment: 31.3, 62.5, 125, 250, 500, 1000 and 2000 μg/mL with and without metabolic activation
Chromosomal aberration test
6-h reatment: 250, 500*, 1000* and 2000* μg/mL with and without metabolic activation
24-h treatment: 62.5, 125, 250*, 354*, 500*, 707, 1000, 1410 and 2000 μg/mL without metabolic activation
The highest dose for short-term treatment was set to be 2000 μg/mL since no cytotoxicity was observed. After 24-h treatment cytotoxicity was observed at 2000 µg/mL. Therefore, 500 µg/mL was selected as the maximum dose (next dose to obtain RPD or RICC > 40% and < 50%)
* chosen for analysis
The concentrations were corrected by the purity of the test substance because the purity was below 95% (50% aqueous solution). - Vehicle / solvent:
- - Vehicle/solvent used: water
- Justification for choice of solvent/vehicle: The test substance was dissolved in distilled water at 200 mg/mL. This solution was considered to be stable based on a lack of change in colour, exothermic reaction and gas generation until 2 h after preparation at room temperature. Therefore, distilled water was selected as a vehicle. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- other: cyclophosphamide monohydrate (CPA): +S9, 4µg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 6 h and 24 h (continuous 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 6 h treatement: 24 h; 24 treatment: 24 h
SPINDLE INHIBITOR (cytogenetic assays): democolcine (10 µg/mL)
STAIN (for cytogenetic assays): Giemsa 2%
NUMBER OF REPLICATIONS: duplicates in one experiment
NUMBER OF CELLS EVALUATED: 300 metaphases per dose (75 cells per specimen) containing 25 ± 2 chromosomes
DETERMINATION OF CYTOTOXICITY
- Method: When RPD or RICC was less than 50%, it was judged that cytotoxicity was defined.
Population doubling (PD), RPD and RICC were calculated by the following formulas:
PD= [log {(Number of cells at the end of the culture)/ (Number of cells at the start of the treatment)}] / log 2
RPD = (PD in test substance group) / (PD in solvent control group) x 100
RICC = [Increase in number of cells in test substance group {(the end of the culture) - (the start of the treatment)}] / [Increase in number of cells in solvent control group {(the end of the culture) - (the start of the treatment)}] x 100
OTHER EXAMINATIONS:
- Determination of polyploidy: yes, cells with triploid or more (38 or more chromosomes)
- Determination of endoreplication: yes, among 300 metaphase cells per dose (75 cells per specimen) - Evaluation criteria:
- The acceptability criteria was considered to be negative, which were either in a) or in b ):
a) all results are inside the distribution of the historical data of the negative control group,
b) outside the distribution of the historical data of the negative control group, but none of the doses of the test substance exhibits a statistically significant increase compared with the concurrent negative control.
Providing that all acceptability criteria in case of c) to e) or f) were fulfilled, a test substance was considered to be positive:
c) outside the distribution of the historical data of the negative control group,
d) the doses of the test substance exhibits a statistically significant increase compared with
the concurrent negative control,
e) the increase of the frequencies of cells with chromosomal aberrations is dose-related,
f) both chromosomal aberration test and confirmation test are fulfilled in case of c) and d). - Statistics:
- Statistical method was performed in order to compare the frequencies of cells with structural aberrations and numerically aberrant cells. Significance level of these tests was both sides of 1% and 5%. Fisher's exact test was performed in order to compare in the solvent control group with positive control group.
Fisher's exact test was performed in order to compare in the solvent control group with test substance group when the frequencies of cells with chromosomal aberrations in each test substance group were outside the distribution of the historical data of the solvent control group. As a result of Fisher's exact test, increased significantly compared to the solvent control group, it was evaluated by using Cochran-Armitage trend test that the dose dependency was exhibited. - Key result
- Species / strain:
- mammalian cell line, other: CHL/IU
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 6-h treatment: +/-S9: no cytotoxicity observed up to 2000 µg/mL; at 24-h treatment: -S9: at and above 500 µg/mL
- 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: No corrosion of the culture dish was observed in any treatment method.
- Precipitation: No precipitation of the test substance in the culture medium was observed.
RANGE-FINDING/SCREENING STUDIES: A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. 2000 µg/mL was applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 31.3 to 2000 µg/mL were chosen for the evaluation of cytotoxicity. No cytotoxicity was observed in "+S9 mix". Therefore, the maximum concentration was selected to be 2000 μg/mL with metablic activation. Cytotoxicity was observed in "-S9 mix". Therefore, the dose which RPD or RICC was 40% or more and 50% or less was selected as the maximum dose.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: RICC or RPD (threshold for cytotoxicity: 50%)
HISTORICAL CONTROL DATA:
+/- S9, 6h and 24 treatment:
Negative control: The frequencies were within the range of the historical data.
Positive control: The frequency was within the range of the historical data. A statistically significant increase was observed at both sides of 1 % level.
Test substance: The frequencies cells with structural and numerical aberrations were well within the range of the historical data of the solvent control. Therefore, it was judged to be negative. - Conclusions:
- Under conditions of the in vitro chromosome aberration test the test substance did not induce chromosome aberrations in CHL/IU cells.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 17 Oct - 28 Nov 2017
- Reliability:
- 1 (reliable without restriction)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- 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:
- Commission Regulation (EC) No 440/2008 of 30 May 2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- OGYÉI, National Institute of Pharmacy and Nutrition, Budapest, Hungary
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Target gene:
- hprt locus
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Remarks:
- K1
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: American Type Culture Collection, Manassas,Virginia, USA
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Ham's F12 medium supplemented with 10% FBS, 0.01 mL/mL L-Glutamine and 0.01 mL/mL antibiotic-antimycotic solution (10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin-B)
- 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 phenobarbital/β-naphthoflavone
- Test concentrations with justification for top dose:
- Preliminary Toxicity Test (5-h treatment)
5-h treatment: 3.906, 7.813, 15.625, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL with and without metabolic activation
24-h treatment: 3.906, 7.813, 15.625, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL without metabolic activation
Assay 1
5-h treatment: 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL with and without metabolic activation
Assay 2
5-h treatment: 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL with metabolic activation
24-h treatment: 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL without metabolic activation - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: The solvent was chosen for its solubility properties, its relative non-toxicity to the cell cultures and its compatibility with the metabolic activation system. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 24 h
- Exposure duration: Assay 1: 5 h with and without metabolic activation; Assay 2: 5 h with metabolic activation and 24 h without metabolic activation
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days
SELECTION AGENT (mutation assays): 10 µg/mL 6-thioguanine (6-TG)
NUMBER OF REPLICATIONS: duplicates (triplicates for survival and viability measurements) each in two experiments
STAINING TECHNIQUE USED: 10% Giemsa
DETERMINATION OF CYTOTOXICITY
- Method: Relative survival was assessed by comparing the cloning efficiency of the treated groups to the vehicle/solvent control.
- OTHER:
Parameters evaluated:
Survival (colony-forming ability at the end of the treatment period), viability (colony-forming ability at the end of the 7 day expression period following the treatment) and mutagenicity (colony-forming ability at the end of the 7-day expression period following the treatment, in the presence of
6-thioguanine as a selective agent) was determined. - Evaluation criteria:
- The assay was considered valid if all the following criteria were met:
1. The mutant frequency in the negative (vehicle/solvent) control cultures was in accordance with the historical control data.
2. The positive control chemicals induced a clear increase in mutant frequency.
3. The cloning efficiency of the negative controls was in the range of 60-140% on Day 1 and 70-130% on Day 8.
4. At least four test item concentrations in duplicate cultures were presented.
The test item was considered to be mutagenic in this assay if the following criteria were met:
1. The assay is valid.
2. The mutant frequency at one or more doses is significantly greater than that of the relevant negative (vehicle) control (p<0.05).
3. Increase of the mutant frequency is reproducible.
4. There is a dose-response relationship. - Statistics:
- The mutation frequencies were statistically analyzed. Statistical evaluation of data was performed with the SPSS PC+4.0 statistical program package (SPSS Hungary Ltd., Budapest, Hungary). The heterogeneity of variance between groups was checked by Bartlett`s test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of not normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test. Data also were checked for a trend in mutation frequency with treatment dose using Microsoft Excel 2010 software (R-squared values were calculated for the log concentration versus the mutation frequency). In the statistical analysis, negative trends were not considered significant.
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- (+S9: 125 µg/mL; -S9: 1000µg/mL)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolarity: There were no large changes in pH and osmolality after treatment in any cases.
- Precipitation:
Assay 1: Insolubility (minimal amount of precipitate) was detected at 125 - 2000 µg/mL concentration range in the final treatment medium at the end of the treatment in the experiments with metabolic activation.
Assay 2: Insolubility (minimal amount of precipitate) was detected at 1000 - 2000 µg/mL concentrations in the final treatment medium at the end of the treatment in the experiments with metabolic activation. The precipitation did not interfere with the reading of the results.
RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity experiment was performed in order to determine the concentration range for the mutagenicity experiments. This experiment was performed in the presence (5 h treatment) and absence (5 h and 24 h treatment) of metabolic activation. Test item concentrations between 3.906 µg/mL and 2000 µg/mL were used. Precipitation was detected at 2000 µg/mL in the preliminary experiment.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Assay 1: In the presence and absence of S9-mix (5-h treatment), no marked cytotoxicity of the test item was observed (the highest concentration of 2000 µg/mL showed a relative survival of 103% and 88% with and without S9-mix, respectively). Therefore, an evaluation was made using data of all seven concentrations.
Assay 2: In the presence (5-h treatment) and absence of S9-mix (24-h treatment), similarly to the first test, no marked cytotoxicity of the test item was observed (the highest concentration of 2000 µg/mL showed a relative survival of 88% and 84% with and without S9-mix, respectively). Therefore, an evaluation was made using data of all seven concentrations. - Conclusions:
- Under the experimental conditions of the gene mutation assay the test item did not induce gene mutations at the HPRT locus in CHO cells with and without metabolic activation.
Referenceopen allclose all
Table 1. Results of experiment 1 (pre-incubation method)
|
Number of revertant colonies per plate |
||||
S9-Mix |
Without |
||||
Test substance (µg/plate) |
TA 100 |
TA 1535 |
WP2 uvr A |
TA 98 |
TA1537 |
SC |
133 ± 10 |
13 ± 3 |
32 ± 7 |
16 ± 2 |
16 ± 3 |
4.88 |
136 ± 17 |
9 ± 5 |
34 ± 8 |
15 ± 4 |
16 ± 1 |
19.5 |
119 ± 7 |
14 ± 4 |
37 ± 8 |
15 ± 2 |
18 ± 8 |
78.1 |
130 ± 15 |
9 ± 2 |
39 ± 2 |
17 ± 3 |
19 ± 7 |
313 |
136 ± 13 |
10 ± 5 |
36 ± 6 |
17 ± 3 |
16 ± 10 |
1250 |
149 ± 4 |
10 ± 4 |
31 ± 9 |
21 ± 4 |
16 ± 3 |
5000 |
132 ± 20 |
16 ± 6 |
34 ± 7 |
18 ± 7 |
15 ± 3 |
Positive Control |
AF-2 |
NaN3 |
AF-2 |
AF-2 |
ICR-191 |
Dose (µg/plate) |
0.01 |
0.5 |
0.01 |
0.1 |
0.5 |
Number of revertant colonies/plate |
899 ± 70 |
361 ± 31 |
164 ± 8 |
478 ± 27 |
720 ± 42 |
S9-Mix |
With |
||||
Test substance (µg/plate) |
TA 100 |
TA 1535 |
WP2 uvr A |
TA 98 |
TA 1537 |
SC |
139 ± 17 |
10 ± 2 |
29 ± 3 |
24 ± 3 |
20 ± 10 |
4.88 |
119 ± 9 |
15 ± 6 |
33 ± 7 |
24 ± 5 |
20 ± 3 |
19.5 |
134 ± 12 |
14 ± 3 |
35 ± 11 |
24 ± 5 |
20 ± 3 |
78.1 |
135 ± 16 |
6 ± 2 |
37 ± 10 |
31 ± 15 |
18 ± 3 |
313 |
126 ± 8 |
12 ± 1 |
34 ± 4 |
27 ± 5 |
17 ± 7 |
1250 |
117 ± 10 |
7 ± 1 |
32 ± 6 |
20 ± 1 |
22 ± 10 |
5000 |
113 ± 17 |
8 ± 4 |
40 ± 2 |
26 ± 6 |
22 ± 5 |
Positive Control |
2AA |
2AA |
2AA |
2AA |
2AA |
Dose (µg/plate) |
1 |
2 |
10 |
0.5 |
2 |
Number of revertant colonies/plate |
1517 ± 53 |
368 ± 11 |
627 ± 56 |
506 ± 26 |
250 ± 35 |
SC = Solvent Control (distilled water) NaN3: sodium acide; 2AA: 2-Aminoanthracene; AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamine |
|||||
Table 2. Results of experiment 2 (pre-incubation method)
|
Number of revertant colonies per plate |
||||
S9-Mix |
Without |
||||
Test substance (µg/plate) |
TA 100 |
TA 1535 |
WP2 uvr A |
TA 98 |
TA1537 |
SC |
131 ± 12 |
13 ± 5 |
38 ± 4 |
18 ± 6 |
22 ± 2 |
313 |
123 ± 26 |
12 ± 1 |
30 ± 8 |
26 ± 8 |
19 ± 2 |
625 |
144 ± 23 |
14 ± 3 |
34 ± 8 |
21 ± 3 |
19 ± 8 |
1250 |
142 ± 20 |
9 ± 2 |
34 ± 10 |
19 ± 4 |
22 ± 3 |
2500 |
143 ± 2 |
9 ± 5 |
29 ± 8 |
20 ± 5 |
18 ± 2 |
5000 |
133 ± 20 |
10 ± 2 |
30 ± 8 |
17 ± 5 |
17 ± 5 |
Positive Control |
AF-2 |
NaN3 |
AF-2 |
AF-2 |
ICR-191 |
Dose (µg/plate) |
0.01 |
0.5 |
0.01 |
0.1 |
0.5 |
Number of revertant colonies/plate |
969 ± 45 |
324 ± 32 |
157 ± 5 |
575 ± 39 |
658 ± 55 |
S9-Mix |
With |
||||
Test substance (µg/plate) |
TA 100 |
TA 1535 |
WP2 uvr A |
TA 98 |
TA 1537 |
SC |
144 ± 8 |
11 ± 3 |
36 ± 7 |
29 ± 11 |
25 ± 0 |
313 |
109 ± 13 |
8 ± 5 |
31 ± 8 |
28 ± 4 |
27 ± 7 |
625 |
123 ± 18 |
9 ± 3 |
33 ± 10 |
30 ± 10 |
27 ± 6 |
1250 |
128 ± 4 |
11 ± 6 |
39 ± 8 |
21 ± 7 |
27 ± 7 |
2500 |
114 ± 5 |
8 ± 2 |
37 ± 3 |
29 ± 4 |
21 ± 6 |
5000 |
114 ± 22 |
9 ± 6 |
35 ± 8 |
26 ± 5 |
26 ± 14 |
Positive Control |
2AA |
2AA |
2AA |
2AA |
2AA |
Dose (µg/plate) |
1 |
2 |
10 |
0.5 |
2 |
Number of revertant colonies/plate |
1757 ± 91 |
385 ± 35 |
585 ± 42 |
485 ± 29 |
274 ± 35 |
SC = Solvent Control (distilled water) NaN3: sodium acide; 2AA: 2-Aminoanthracene; AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamine |
|||||
Table 3. Histrorical negative control data (mean ± 3S.D.)
-S9 mix |
+S9mix |
|||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|
Mean S.D. |
119 25 |
11 4 |
32 10 |
20 6 |
12 6 |
124 23 |
11 4 |
31 10 |
29 8 |
14 6 |
UpperLimit Lower Limit |
194 44 |
23 1 |
62 2 |
38 2 |
30 1 |
193 55 |
23 1 |
61 1 |
53 5 |
32 1 |
Table 4. Historical positive control data (mean± 3S.D.)
|
-S9 mix |
+S9mix |
||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|
Chemical |
AF-2 |
NaN3 |
AF-2 |
AF-2 |
ICR-191 |
2AA |
2AA |
2AA |
2AA |
2AA |
Dose(µg/plate) |
0.01 |
0.5 |
0.01 |
0.1 |
0.5 |
1 |
2 |
10 |
0.5 |
2 |
Mean S.D. |
854 104 |
312 57 |
112 21 |
498 54 |
635 103 |
1561 194 |
292 35 |
379 101 |
397 40 |
207 39 |
Upper Limit Lower Limit |
1166 542 |
483 141 |
175 49 |
660 336 |
944 326 |
2143 979 |
397 187 |
682 76 |
517 277 |
324 90 |
AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide
NaN3: Sodiumazide
2AA.: 2-Aminoanthracene
Test period: From September, 2016 to February, 2017
Table 1: Results of 6h treatment experiment.
Test substance |
Concentration (µg/mL) |
Total number of cells with structural aberrations (% of 300 cells counted) |
Number of gaps (frequency %) |
Cell growth rate (%) |
RPD (%) |
RICC (%) |
Total number of cells with numerical aberrations (% of 300 cells counted) |
Without metabolic activation |
|||||||
Solvent (water) |
- |
2 |
0 |
100 |
100 |
100 |
0 |
0 |
1 |
1 |
|||||
2 (0.7) |
1 (0.3) |
1 (0.3) |
|||||
MMC |
0.05 |
37 |
0 |
84.5 |
75.7 |
69.0 |
0 |
40 |
0 |
73.6 |
55.7 |
47.1 |
1 |
||
77 (25.7)** |
0 (0.0) |
79.1 |
65.7 |
58.1 |
1 (0.3) |
||
Test substance |
250 |
- |
100.1 |
100.2 |
100.2 |
- |
|
94.9 |
92.6 |
89.9 |
|||||
97.5 |
96.4 |
95.1 |
|||||
500 |
1 |
0 |
94.3 |
91.6 |
88.6 |
0 |
|
0 |
0 |
91.0 |
86.4 |
82.0 |
0 |
||
1 (0.3) |
0 (0.0) |
92.7 |
89.0 |
85.3 |
0 (0.0) |
||
1000 |
2 |
0 |
90.2 |
85.1 |
80.3 |
1 |
|
0 |
0 |
94.5 |
91.9 |
89.0 |
0 |
||
2 (0.7) |
0 (0.0) |
92.4 |
88.5 |
84.7 |
0 (0.3) |
||
2000 |
1 |
1 |
77.8 |
63.7 |
55.5 |
0 |
|
2 |
0 |
69.3 |
47.0 |
38.5 |
0 |
||
3 (1.0) |
1 (0.3) |
73.6 |
55.4 |
47.0 |
0 (0.0) |
||
With metabolic activation |
|||||||
Solvent (water) |
- |
1 |
0 |
100 |
100 |
100 |
0 |
2 |
0 |
1 |
|||||
3 (1.0) |
0 (0.0) |
1 (0.3) |
|||||
CPA |
4 |
27 |
0 |
84.5 |
75.7 |
69.0 |
0 |
40 |
0 |
73.6 |
55.7 |
47.1 |
1 |
||
77 (25.7)** |
0 (0.0) |
79.1 |
65.7 |
58.1 |
1 (0.3) |
||
Test substance |
250 |
- |
102.3 |
103.3 |
104.5 |
- |
|
102.5 |
103.6 |
105.0 |
|||||
102.4 |
103.5 |
104.8 |
|||||
500 |
1 |
0 |
95.9 |
93.9 |
91.7 |
0 |
|
1 |
0 |
97.3 |
96.1 |
94.6 |
3 |
||
2 (0.7) |
0 (0.0) |
96.6 |
95.0 |
93.2 |
3 (1.0) |
||
1000 |
3 |
0 |
94.4 |
91.7 |
88.8 |
1 |
|
2 |
0 |
89.3 |
83.6 |
78.5 |
0 |
||
5 (1.7) |
0 (0.0) |
91.9 |
87.7 |
83.7 |
1 (0.3) |
||
2000 |
1 |
0 |
81.4 |
70.3 |
62.8 |
0 |
|
2 |
0 |
77.7 |
63.6 |
55.4 |
0 |
||
3 (1.0) |
0 (0.0) |
79.6 |
67.0 |
59.1 |
0 (0.0) |
||
The number of aberrant cells at each dish is shown at the first and the second lines. The total number of them is shown at the third line.
Cell growth rate, RPD and RICC at each dish is shown at the first and the second lines. The average of them is shown at the third line.
RPD: relative population doubling, RICC: relative increase in cell count, MMC: mitomycin C, CPA: cyclophosphamide monohydrate
In all treatment methods, the specimens at 250 µg/mL were not observed.
**: significant increase compared to solvent control at p<0.01 [Fisher's exact test]
Table 2. Results of 24 h continuous treatment
Test substance |
Concentration (µg/mL) |
Total number of cells with structural aberrations |
Number of gaps (frequency %) |
Cell growth rate (%) |
RPD (%) |
RICC (%) |
Total number of cells with numerical aberrations |
Without metabolic activation |
|||||||
Solvent (water) |
- |
1 |
0 |
100 |
100 |
100 |
0 |
1 |
1 |
0 |
|||||
2 (0.7) |
1 (0.3) |
0 (0.0) |
|||||
MMC |
0.05 |
82 |
1 |
78.0 |
69.8 |
60.7 |
0 |
94 |
1 |
77.1 |
68.4 |
59.1 |
0 |
||
176 (58.7)** |
2 (0.7) |
77.6 |
69.1 |
59.9 |
0 (0.0) |
||
Test substance |
62.5 |
- |
91.2 |
88.8 |
84.3 |
- |
|
98.1 |
97.7 |
96.6 |
|||||
94.7 |
93.3 |
90.5 |
|||||
125 |
- |
90.1 |
87.4 |
82.4 |
- |
||
92.5 |
90.5 |
86.6 |
|||||
91.3 |
89.0 |
84.5 |
|||||
250 |
2 |
0 |
81.6 |
75.3 |
67.2 |
0 |
|
5 |
0 |
84.5 |
79.5 |
72.4 |
0 |
||
7 (2.3) |
0 (0.0) |
83.1 |
77.4 |
69.8 |
0 (0.0) |
||
354 |
2 |
1 |
72.9 |
61.6 |
51.7 |
0 |
|
2 |
1 |
74.9 |
64.9 |
55.3 |
0 |
||
4 (1.3) |
2 (0.7) |
73.9 |
63.3 |
53.5 |
0 (0.0) |
||
500 |
3 |
0 |
64.6 |
46.8 |
36.8 |
0 |
|
1 |
0 |
66.8 |
50.9 |
40.7 |
0 |
||
4 (1.3) |
0 (0.0) |
65.7 |
48.9 |
38.8 |
0 (0.0) |
||
707 |
- |
62.6 |
43.0 |
33.3 |
- |
||
62.8 |
43.4 |
33.6 |
|||||
62.7 |
43.2 |
33.5 |
|||||
1000 |
n.s. |
56.7 |
30.8 |
22.6 |
n.s. |
||
55.2 |
27.7 |
20.0 |
|||||
56.0 |
29.3 |
21.3 |
|||||
1410 |
n.s. |
51.2 |
18.6 |
12.9 |
n.s. |
||
48.1 |
11.0 |
7.4 |
|||||
49.7 |
14.8 |
10.2 |
|||||
2000 |
n.s. |
|
-7.2 |
-4.5 |
n.s. |
||
|
-9.9 |
-6.1 |
|||||
|
-8.6 |
-5.3 |
|||||
The number of aberrant cells at each dish is shown at the first and the second lines. The total number of them is shown at the third line.
Cell growth rate, RPD and RICC at each dish is shown at the first and the second lines. The average of them is shown at the third line.
RPD: relative population doubling, RICC: relative increase in cell count, MMC: mitomycin C, n.s.: no specimens
The specimens at 62.5, 125 and 707 µg/mL were not observed.
**: significant increase compared to solvent control at p<0.01 [Fisher's exact test]
Tabel 3. Historical negative control data
Treatment method |
Frequency of cells with chromosomal aberrations (%) |
|||
Structural aberration |
Numerical aberration |
|||
Mean |
SD |
Mean |
SD |
|
Short-term treatment, without S9 mix |
1.9 |
0.54 |
0.4 |
0.31 |
Short-term treatment, with S9mix |
1.5 |
0.65 |
0.5 |
0.38 |
24 h continuous treatment |
1.5 |
0.52 |
0.4 |
0.33 |
Treatment method |
Range of frequency of cells with chromosomal aberrations (%, values in parentheses indicate 95% control limit) |
||||
Structural aberration |
Numerical aberration |
||||
Lowercontrol limit |
Upper control limit |
Lower control limit |
Upper control limit |
||
Short-termtreatment |
Without S9mix |
<0 (<0) |
4.3 (4.2) |
<0 (<0) |
1.6 (1.5) |
With S9mix |
<0 (< 0) |
3.7 (3.6) |
<0 (<0) |
1.8 (1.7) |
|
24 h continuous treatment |
<0 (< 0) |
3.6 (3.5) |
<0 (<0) |
1.6 (1.5) |
|
The minimum range below 0 was shown "<0".
Tabel 4. Historical positive control data
Treatment method |
Substance |
Dose (µg/mL) |
Frequency of cells with chromosomal aberrations (%) |
|
Structural aberration |
||||
Mean |
SD |
|||
Short-term treatment, without S9mix |
MMC |
0.05 |
28.8 |
3.50 |
Short-term treatment, with S9mix |
CPA |
4.00 |
32.2 |
3.41 |
24 h continuous treatment |
MMC |
0.05 |
64.0 |
3.05 |
The minimum range below 0 was shown "<0".
The latest 20 test data completed by February, 2017 were used.
Upper and lower control limits were calculated from number of cells with chromosomal aberrations using c chart. The value was converted to the frequency from the number of cells with chromosomal aberrations.
Table 1. Summary of survival results (colony-forming ability at the end of the treatment period)
| S9 mix | Treatment period (hours) | Study phase | Test item or control concentration | Total | Cloning | Relative |
| number | Efficiency | Survival (%) | ||||
| of colonies | (CE) | on plates | ||||
| + | 5 | Assay 1 | 2000 µg/mL | 1178 | 0.982 | 103 |
| 1000 µg/mL | 1006 | 0.838 | 88 | |||
| 500 µg/mL | 1082 | 0.902 | 95 | |||
| 250 µg/mL | 1100 | 0.917 | 96 | |||
| 125 µg/mL | 1159 | 0.966 | 101 | |||
| 62.5 µg/mL | 1297 | 1.081 | 113 | |||
| 31.25 µg/mL | 1255 | 1.046 | 110 | |||
| Negative control | 1144 | 0.953 | 100 | |||
| Vehicle control for DMBA (DMSO) | 1090 | 0.908 | 95 | |||
| Untreated control | 1075 | 0.896 | 94 | |||
| Positive control (DMBA) | 38 | 0.032 | 3 | |||
| - | 5 | Assay 1 | 2000 µg/mL | 1113 | 0.928 | 88 |
| 1000 µg/mL | 1261 | 1.051 | 99 | |||
| 500 µg/mL | 1049 | 0.874 | 83 | |||
| 250 µg/mL | 1143 | 0.953 | 90 | |||
| 125 µg/mL | 1312 | 1.093 | 103 | |||
| 62.5 µg/mL | 1270 | 1.058 | 100 | |||
| 31.25 µg/mL | 1200 | 1 | 94 | |||
| Vehicle control | 1271 | 1.059 | 100 | |||
| Vehicle control for EMS (DMSO) | 1189 | 0.991 | 94 | |||
| Untreated control | 1113 | 0.928 | 88 | |||
| Positive control (EMS) | 893 | 0.744 | 70 | |||
| + | 5 | Assay 2 | 2000 µg/mL | 916 | 0.763 | 88 |
| 1000 µg/mL | 1188 | 0.99 | 114 | |||
| 500 µg/mL | 984 | 0.82 | 94 | |||
| 250 µg/mL | 1180 | 0.983 | 113 | |||
| 125 µg/mL | 1076 | 0.897 | 103 | |||
| 62.5 µg/mL | 1048 | 0.873 | 100 | |||
| 31.25 µg/mL | 1296 | 1.08 | 124 | |||
| Vehicle control | 1044 | 0.87 | 100 | |||
| Vehicle control for DMBA (DMSO) | 1056 | 0.88 | 101 | |||
| Untreated control | 1288 | 1.073 | 123 | |||
| Positive control (DMBA) | 27 | 0.023 | 3 | |||
| - | 24 | Assay 2 | 2000 µg/mL | 888 | 0.74 | 84 |
| 1000 µg/mL | 1132 | 0.943 | 107 | |||
| 500 µg/mL | 1232 | 0.027 | 117 | |||
| 250 µg/mL | 1084 | 0.903 | 103 | |||
| 125 µg/mL | 1180 | 0.983 | 112 | |||
| 62.5 µg/mL | 1032 | 0.86 | 98 | |||
| 31.25 µg/mL | 1268 | 1.057 | 120 | |||
| Vehicle control | 1056 | 0.88 | 100 | |||
| Vehicle control for EMS (DMSO) | 1004 | 0.837 | 95 | |||
| Untreated control | 1168 | 0.973 | 111 | |||
| Positive control (EMS) | 588 | 0.49 | 56 |
Results of 3 paralell dishes.
DMBA = 7,12-Dimethylbenz[a]anthracene, 15 µg/mL
EMS = Ethyl methanesulfonate, 0.4 µL/mL
Vehicle control = Distilled water
DMSO = Dimethyl sulfoxide
Table 2. Summary of viability results (colony-forming ability at the end of the 7 day expression period following the treatment)
| S9 mix | Treatment period (hours) | Study phase | Test item or control concentration | Total number of colonies | Cloning Efficiency |
| (CE) | |||||
| + | 5 | Assay 1 | 2000 µg/mL | 952 | 0.793 |
| 1000 µg/mL | 964 | 0.803 | |||
| 500 µg/mL | 1088 | 0.907 | |||
| 250 µg/mL | 1071 | 0.893 | |||
| 125 µg/mL | 1112 | 0.927 | |||
| 62.5 µg/mL | 1021 | 0.851 | |||
| 31.25 µg/mL | 1077 | 0.898 | |||
| Vehicle control | 1158 | 0.965 | |||
| Vehicle control for DMBA (DMSO) | 1046 | 0.872 | |||
| Untreated control | 1053 | 0.878 | |||
| Positive control (DMBA) | 1057 | 0.881 | |||
| - | 5 | Assay 1 | 2000 µg/mL | 1029 | 0.858 |
| 1000 µg/mL | 1113 | 0.928 | |||
| 500 µg/mL | 948 | 0.79 | |||
| 250 µg/mL | 943 | 0.786 | |||
| 125 µg/mL | 953 | 0.794 | |||
| 62.5 µg/mL | 1003 | 0.836 | |||
| 31.25 µg/mL | 1098 | 0.915 | |||
| Vehicle control | 1143 | 0.953 | |||
| Vehicle control for EMS (DMSO) | 1160 | 0.967 | |||
| Untreated control | 1039 | 0.866 | |||
| Positive control (EMS) | 797 | 0.664 | |||
| + | 5 | Assay 2 | 2000 µg/mL | 1193 | 0.994 |
| 1000 µg/mL | 1225 | 1.021 | |||
| 500 µg/mL | 1180 | 0.983 | |||
| 250 µg/mL | 1087 | 0.906 | |||
| 125 µg/mL | 1089 | 0.908 | |||
| 62.5 µg/mL | 1121 | 0.934 | |||
| 31.25 µg/mL | 1193 | 0.994 | |||
| Vehicle control | 1237 | 1.031 | |||
| Vehicle control for DMBA (DMSO) | 1170 | 0.975 | |||
| Untreated control | 1289 | 1.074 | |||
| Positive control (DMBA) | 1224 | 1.02 | |||
| - | 24 | Assay 2 | 2000 µg/mL | 1142 | 0.952 |
| 1000 µg/mL | 1198 | 0.998 | |||
| 500 µg/mL | 1132 | 0.943 | |||
| 250 µg/mL | 1274 | 1.062 | |||
| 125 µg/mL | 1150 | 0.958 | |||
| 62.5 µg/mL | 1167 | 0.973 | |||
| 31.25 µg/mL | 1120 | 0.933 | |||
| Vehicle control | 1157 | 0.964 | |||
| Vehicle control for EMS (DMSO) | 1185 | 0.988 | |||
| Untreated control | 1028 | 0.857 | |||
| Positive control (EMS) | 586 | 0.488 |
Results of 3 parallel dishes.
DMBA = 7,12-Dimethylbenz[a]anthracene, 15 µg/mL
EMS = Ethyl methanesulfonate, 0.4 µL/mL
Vehicle control = Distilled water
DMSO = Dimethyl sulfoxide
Table 3. Summary of mutagenicity results (colony forming ability at the end of selection period)
| S9 mix | Treatment period (hours) | Study phase | Test item or control concentration | Total number of colonies | Mutant frequency |
| + | 5 | Assay 1 | 2000 µg/mL | 25 | 8.2 |
| 1000 µg/mL | 21 | 6.5 | |||
| 500 µg/mL | 23 | 6.4 | |||
| 250 µg/mL | 14 | 3.9 | |||
| 125 µg/mL | 16 | 4.3 | |||
| 62.5 µg/mL | 20 | 5.9 | |||
| 31.25 µg/mL | 21 | 5.9 | |||
| Vehicle control | 21 | 5.5 | |||
| Vehicle control for DMBA (DMSO) | 19 | 5.5 | |||
| Untreated control | 20 | 5.7 | |||
| Positive control (DMBA) | 1022 | 293.3** | |||
| - | 5 | Assay 1 | 2000 µg/mL | 17 | 5 |
| 1000 µg/mL | 18 | 5 | |||
| 500 µg/mL | 16 | 5.1 | |||
| 250 µg/mL | 19 | 6.1 | |||
| 125 µg/mL | 17 | 5.3 | |||
| 62.5 µg/mL | 19 | 5.7 | |||
| 31.25 µg/mL | 13 | 3.7 | |||
| Vehicle control | 23 | 6 | |||
| Vehicle control for EMS (DMSO) | 20 | 5.2 | |||
| Untreated control | 21 | 6.1 | |||
| Positive control (EMS) | 740 | 279.4** | |||
| + | 5 | Assay 2 | 2000 µg/mL | 21 | 5.3 |
| 1000 µg/mL | 23 | 5.6 | |||
| 500 µg/mL | 27 | 6.9 | |||
| 250 µg/mL | 19 | 5.2 | |||
| 125 µg/mL | 17 | 4.7 | |||
| 62.5 µg/mL | 21 | 5.7 | |||
| 31.25 µg/mL | 19 | 4.8 | |||
| Vehicle control | 20 | 4.9 | |||
| Vehicle control for DMBA (DMSO) | 23 | 5.9 | |||
| Untreated control | 17 | 4 | |||
| Positive control (DMBA) | 1445 | 353.6** | |||
| - | 24 | Assay 2 | 2000 µg/mL | 30 | 7.9** |
| 1000 µg/mL | 22 | 5.6 | |||
| 500 µg/mL | 19 | 5.1 | |||
| 250 µg/mL | 21 | 5 | |||
| 125 µg/mL | 19 | 4.9 | |||
| 62.5 µg/mL | 16 | 4.1 | |||
| 31.25 µg/mL | 19 | 5.2 | |||
| Vehicle control | 18 | 4.7 | |||
| Vehicle control for EMS (DMSO) | 20 | 5.1 | |||
| Untreated control | 18 | 5.3 | |||
| Positive control (EMS) | 1399 | 722.6** |
** = Statistically significant increase (at p< 0.01) compared to the relevant vehicle control
Mutant frequencies refer to 10E06 clonable cells.
DMBA = 7,12-Dimethylbenz[a]anthracene, 15 µg/mL
EMS = Ethyl methanesulfonate, 0.4 µL/mL
Vehicle control = Distilled water
DMSO = Dimethyl sulfoxide
Statistically significant increase in mutation frequency (at p<0.01 level) was observed in assay 2 in the absence of metabolic activation at 2000 μg/mL concentration (see Table 3), although the observed value was within the general historical control range. Furthermore, the observed mutant frequencies (7.9 x 10-6) was within the expected range of the negative control samples according to the relevant OECD guideline (expected range: 5-20 x 10- 6). No dose response to the treatment was observed (a trend analysis showed no effect of treatment). The negative result was confirmed by the Assay 1 without metabolic activation.
Table 4. Historical control data (updated 17 Oct 2017)
| Mutation frequency | |||
| (Number of 6-TG resistant mutants per 106clonable cells) | |||
| Untreated control | |||
| 5-hour, S9+ | 5-hour, S9- | 24-hour, S9- | |
| mean | 18.3 | 20.7 | 19 |
| standard deviation | 15.1 | 16.4 | 17.2 |
| minimum | 5.1 | 5.5 | 3.3 |
| maximum | 64.1 | 55.5 | 58 |
| n | 27 | 13 | 14 |
| DMSO control | |||
| 5-hour, S9+ | 5-hour, S9- | 24-hour, S9- | |
| mean | 21.8 | 18.9 | 18.4 |
| standard deviation | 15.9 | 11.6 | 14.4 |
| minimum | 5.4 | 6.5 | 6.8 |
| maximum | 57.3 | 47.4 | 48.5 |
| n | 29 | 13 | 14 |
| Distilled water / Water based vehicle control | |||
| mean | 11.5 | 9.1 | 15.5 |
| standard deviation | 3.8 | 3.4 | 5.6 |
| minimum | 6.1 | 5.2 | 9.2 |
| maximum | 15.8 | 11.6 | 20.1 |
| n | 6 | 3 | 3 |
| Positive controls | |||
| DMBA | EMS | EMS | |
| 5-hour, S9+ | 5-hour, S9- | 24-hour, S9- | |
| mean | 905.2 | 445.6 | 1176.6 |
| standard deviation | 562.7 | 118.6 | 610.9 |
| minimum | 141.2 | 239.6 | 363.1 |
| maximum | 2119.4 | 636.6 | 2449.8 |
| n | 27 | 13 | 14 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Gene mutation in bacteria
A bacterial gene mutation assay with the test substance was performed in accordance with OECD guideline 471 and in compliance with GLP (Ogura, 2017). In two independent experiments, the Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537, and the Escherichia coli strain WP2 uvrA were exposed to the test substance using the pre-incubation method, with and without metabolic activation. The test substance was provided in a 50% aqueous solution, but was corrected by the purity before preparation of dosing solutions. Test substance concentrations of 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate were selected for the incubation in the first experiment and 315, 625, 1250, 2500 and 5000 µg/plate for the second experiment. The test substance showed no bacterial toxicity at any dose in the experiments with or without metabolic activation. No biologically relevant increase in revertant numbers was observed after treatment with the test substance in any bacterial strain and at any concentration tested in the presence and absence of metabolic activation. The revertant frequencies of the vehicle control were within the expected range and the positive control chemicals induced marked increases in revertant colonies, demonstrating the effective performance of the experiments. The results of the solvent and positive control fell within the historical control data range. Under the conditions of this experiment, the test substance did not cause mutagenicity in the selected S. typhimurium and E. coli strains in the presence and absence of metabolic activation.
In vitro cytogenicity in mammalian cells
The clastogenic activity of the test substance was investigated in an in vitro mammalian chromosome aberration test in cultured Chinese hamster lung fibroblasts (CHL/IU) performed according to OECD guideline 473 and GLP (Ogura, 2017). The test substance was provided in a 50% aqueous solution, but was corrected by the purity before preparation of dosing solutions. Based on the results of a preliminary cell growth inhibition test, cells were treated with the test substance at 500 - 2000 µg/mL in distilled water for 6 h with and without metabolic activation (short-term treatment) or at concentrations of 250 - 2000 µg/mL for 24 h without metabolic activation (continuous treatment). The chromosomes were prepared 24 h after start of treatment with the test substance. In each experimental group two parallel cultures were analysed. Cells were counted and relative population doubling (RPD) and relative increase in cell count (RICC) was determined. A total of 300 metaphase cells per dose were evaluated for structural and numerical chromosomal aberrations. In the presence of metabolic activation, no cytotoxicity was observed up to the highest evaluated concentration. In the absence of metabolic activation cytotoxicity (threshold: RPD < 50%) was observed at 2000 µg/mL after short term treatment and at 500 µg/mL and above after 24 h treatment. The frequencies of cells with structural aberrations and numerically aberrant cells at all observation doses of the test substance were within the range of the historical data of the solvent control. Mitomycin C (0.05 μg/mL, -S9) and cyclophosphamide monohydrate (4 μg/mL, +S9) were used as positive controls and induced distinct increases in cells with structural chromosome aberrations. No precipitation of the test substance in the culture medium was observed. In conclusion, the test substance did not induce structural chromosomal aberrations in CHO/IU cells in vitro under the experimental conditions reported. Therefore, the test substance is not considered to be clastogenic in this chromosome aberration test, when tested up to the highest evaluable concentrations.
In vitro gene mutation in mammalian cells
The mutagenic activity of the test substance was evaluated in an in vitro mammalian cell gene mutation test according to OECD guideline 476 and in compliance with GLP (Kovács, 2018). The test substance was assessed for its potential to induce gene mutations at the HPRT locus using CHO K1 cells of the Chinese hamster. The study was performed in two independent experiments, using identical experimental procedures. Based on the results of a pre-test, cells were exposed for 5 and 24 h to the test substance up to concentrations of 2000 µg/mL in the absence and presence of metabolic activation. Minimal precipitation of the test substance was detected at 125 - 2000 µg/mL (Experiment 1) and 1000 - 2000 µg/mL (Experiment 2) in the final treatment medium at the end of the treatment in the experiments with metabolic activation. The precipitation did not interfere with the reading of the results. In both experiments, in the presence and absence of metabolic activation no marked cytotoxicity was observed up to the highest concentration of 2000 µg/mL. In Experiment 1, no statistically significant increases in the mutation frequency were observed with and without metabolic activation at any examined concentrations when compared to the vehicle control data. In Experiment 2, statistically significant increase (at p < 0.01 level) was observed at 2000 µg/mL in the absence of metabolic activation. However, the observed value was within the general historical control range. Furthermore, the observed mutant frequencies were within the expected range of the negative control samples according to the relevant OECD guideline. Therefore, this is not considered to indicate a mutagenic effect. With metabolic activation, no statistically significant increases in the mutation frequency were observed at any concentrations when compared with the vehicle control data. The spontaneous mutation frequency of the vehicle control fell within the historical control data range in all assays. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies, showing the sensitivity of the test system and the activity of the metabolic activation system. In conclusion the test substance did not induce gene mutations at the HPRT locus in CHO K1 cells under the experimental conditions reported. Therefore, the test substance is considered to be non-mutagenic in this HPRT test.
Justification for classification or non-classification
The available data on genetic toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.
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