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EC number: 665-911-3 | CAS number: 5855-23-2
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 22 Feb - 04 Mar 2013
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- - As the outcome of this Ames Test is negative, a second confirmatory experiment is recommended (OECD 471). The preliminary test conducted here does not entirely fulfill this requirement, as it was performed according to the same protocol as the main experiment, and as other test concentrations were used. - Duplicates instead of triplicates were used in both experiments without provision of a scientific justification.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 013
- Report date:
- 2013
Materials and methods
Test guideline
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 1997
- Deviations:
- yes
- Remarks:
- - 2-Aminoanthracene was used as the sole indicator for the efficacy of the S9-mix
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- 1-(2,2,4-trimethyl-1,2-dihydroquinolin-1-yl)ethan-1-one
- EC Number:
- 665-911-3
- Cas Number:
- 5855-23-2
- Molecular formula:
- C14 H17 N O
- IUPAC Name:
- 1-(2,2,4-trimethyl-1,2-dihydroquinolin-1-yl)ethan-1-one
Constituent 1
Method
- Target gene:
- his operon (for S. typhimurium strains)
trp operon (for the E.coli strain)
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- other: tested for the presence / absence of amino acid requirements, sensitivity to UV radiation, membrane mutation rfa and the drug resistance factor R-factor
- Species / strain / cell type:
- E. coli WP2 uvr A
- Additional strain / cell type characteristics:
- other: tested for the presence / absence of amino acid requirements, sensitivity to UV radiation, membrane mutation rfa and the drug resistance factor R-factor
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9: Oriental Yeast Co., Ltd. (Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male SD rats treated with Phenobarbital and 5,6-Benzoflavone)
- method of preparation of S9 mix: S9 Mix was prepared at the time of use, and stored by ice cooling during use. Composition: S9: 0.1 mL; MgCl2: 8 µmol; KCl: 33 µmol; Glucose-6-phospate: 5 µmol; NADPH: 4 µmol; NADH: 4 µmol; Na-phosphate buffer (pH 7.4): 100 µmol
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL S9 mix (0.05 mL S9) / 2.7 mL - Test concentrations with justification for top dose:
- The doses were chosen based on the results of a dose-setting study carried out using each of the bacterial strains, with a maximum dose of 5000 µg/plate, and serial dilution by a factor of 4 to give a total of 8 doses. Based on the effects in inhibiting growth of each bacterial strain, the doses below were set in the main study:
- TA100, TA1535, WP2 uvrA, TA98, TA1537 under conditions of S9(-): 4.9, 9.8, 20, 39, 78, 156, 313 µg/plate
- TA100, WP2 uvrA, TA98, TA1537 under conditions of S9(+): 9.8, 20, 39, 78, 156, 313 µg/plate
- TA1535 under conditions of S9(+): 4.9, 9.8, 20, 39, 78, 156, 313 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The solubility of the test substance in water is <50 mg/mL, but solubility in DMSO is ≥ 50 mg/mL and it was stable for 24 hours in a 5% solution at room temperature.
Controls
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- other:
- Remarks:
- 1) TA 100 0.01 µg/plate, TA 98 0.1 µg/plate, WP2 uvrA 0.01 µg/plate (-S9);
2) TA 1537 1.0 µg/plate (-S9);
3) TA 100 1.0 µg/plate, TA 1535 2.0 µg/plate, WP2 uvrA 10.0 µg/plate, TA 98 0.5 µg/plate, TA 1537 2.0 µg/plate (+S9)
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): 2 plates per dose for treated groups and pos. control; 3 plates for neg. control
- Number of independent experiments: 1
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: The bacterial concentrations were measured using as an indicator the absorbance of the culture solutions of each of the strains after 10-fold dilution with physiological saline using a spectrophotometer (measurement wavelength 660 nm, light path length 10 mm):
Strain TA100 TA1535 WP2 uvrA TA98 TA1537
Dose-setting study 3.4 4.7 6.1 3.5 2.8
Main study 3.6 4.6 7.3 3.5 2.8
[Figures indicating viable bacterial count (×10^9/mL)]
- Test substance added: 0.1 mL/plate; preincubation
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min at 37 °C
- Exposure duration/duration of treatment: 48 h
METHODS FOR MEASUREMENTS OF CYTOTOXICITY
- Method: decrease in the number of revertant colonies per plate
METHODS FOR MEASUREMENTS OF GENOTOXICITY
- Colony counting carried out using an automatic colony counter (CA-11 Automatic Colony Analyzer, System Science KK) with surface area correction and correction for counting errors by computer. When the number of colonies as the result of counting with the automatic colony counter is ≥ 1500, the precision of the automatic colony counter falls and so manual counting was carried out with a manual colony counter (manual colony counter No.3833, ANDERMAN & Co., Ltd.). Manual counting was also carried out when counting with the automatic counter was not possible because the colonies were very small due to strong growth inhibition. When the number of colonies was less than 300 with manual counting, all of these were counted and no correction was applied. When the number of colonies was ≥ 300, the plate was divided radially into 8 equal segments; 2 segments were counted and this number, corrected for surface area, was used. - Evaluation criteria:
- Based on the results of the dose-setting study and the main study, an increase of ≥ 2 times in the revertant colony count in the groups treated with the test substance compared with the negative control group was assessed as a positive result when dose-dependence and reproducibility were observed. All other cases were assessed as negative. When a positive result was obtained, a specific activity value was calculated.
- Statistics:
- Statistical analysis was not used in evaluating the results of the study.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- S9(-/+): 313 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- S9(-/+): 313 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- S9(-/+): 313 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- S9(-): 156, 313 µg/plate S9(+) 313 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- S9(-): 156, 313 µg/plate S9(+): 313 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test substance does not dissolve in water at 50 mg/mL or higher.
- Precipitation: Precipitation of the test substance (white) was noted in the S9(±) 5000 µg/plate in the dose-setting study. In the main experiment, no precipitation occurred.
- Contamination: The test without bacteria confirmed the absence of other contaminating microorganisms in the experiment.
RANGE-FINDING/SCREENING STUDIES (Table 1)
A dose-setting study was carried out using each of the bacterial strains, with a maximum dose of 5000 µg/plate, and serial dilutions by a factor of 4, to give a total of 8 doses (0.31, 1.2, 4.9, 20, 78, 313, 1250, 5000 µg/plate). As a result, no increase of 2-fold or greater in revertant colonies was observed compared with the negative control, irrespective of the presence or absence of metabolic activation, for any of the strains. It should be noted that growth inhibition of each of the strains by this test substance was noted at the doses 313 - 5000 µg/plate for all strains with and without metabolic activation. Precipitation of the test substance (white) was noted in the S9(±) 5000 µg/plate.
STUDY RESULTS
No increase of 2-fold or greater in revertant colonies compared with the negative control was noted, irrespective of the presence or absence of metabolic activation for any of the strains, and also no dose-dependent increases were seen. It should be noted that, growth inhibition of each of the strains by this test substance was noted (see *-indicated results in table 2), irrespective of the presence or absence of metabolic activation; precipitation of the test substance was not observed.
HISTORICAL CONTROL DATA
- Positive historical control data: given in table 3; the positive controls induced revertant colonies more than twice the negative control for each strain, confirming validity of the response.
- Negative (solvent/vehicle) historical control data: given in table 3; in all of the tests in the dose-setting study and the main study, the negative control was within the permitted range of the background data for this institution (mean ± 2.5 SD).
Any other information on results incl. tables
Table 1: Test results of the dose-setting study
Metabolic activation Y/N | Dose of test substance (μg/plate) | Revertant colonies/plate | |||||
Base pair substitution | Frame shift | ||||||
TA 100 | TA 1535 | WP2 uvrA | TA 98 | TA 1537 | |||
S9 mix (–) | Negative control | 96 97 104 (99) | 13 14 10 (12) | 33 34 36 (34) | 19 27 21 (19) | 4 5 4 (4) | |
0.31 | 99 98 (99) | 12 12 (12) | 28 33 (31) | 15 18 (17) | 4 4 (4) | ||
1.2 | 96 111 (104) | 8 10 (9) | 32 32 (32) | 16 15 (16) | 3 3 (3) | ||
4.9 | 101 97 (99) | 8 11 (10) | 32 39 (36) | 14 13 (14) | 5 3 (4) | ||
20 | 104 92 (98) | 10 13 (12) | 35 39 (37) | 13 13 (13) | 3 6 (5) | ||
78 | 107 96 (102) | 8 9 (9) | 38 38 (38) | 12 13 (13) | 3 4 (4) | ||
313 | 54* 0* (27) | 0* 0* 0 | 0* 9* (5) | 9* 0* (5) | 0* 0* (0) | ||
1250 | 0* 0* (0) | 0* 0* (0) | 0* 0* (0) | 0* 0* (0) | 0* 0* (0) | ||
5000† | 0* 0* (0) | 0* 0* (0) | 0* 0* (0) | 0* 0* (0) | 0* 0* (0) | ||
S9 mix (+) | Negative control | 128 118 118 (121) | 9 10 10 (10) | 30 38 41 (36) | 19 22 21 (21) | 17 17 23 (19) | |
0.31 | 116 99 (108) | 10 6 (8) | 32 35 (34) | 22 22 (22) | 19 17 (18) | ||
1.2 | 113 87 (100) | 10 7 (9) | 39 32 (36) | 27 29 (28) | 15 16 (16) | ||
4.9 | 95 116 (106) | 7 7 (7) | 30 38 (34) | 23 29 (26) | 14 14 (14) | ||
20 | 99 99 (99) | 6 7 (7) | 33 33 (33) | 20 24 (22) | 15 16 (16) | ||
78 | 93 116 (105) | 7 7 (7) | 26 28 (27) | 19 17 (18) | 16 19 (18) | ||
313 | 77* 69* (73) | 0* 1* (1) | 16* 13* (15) | 11* 7* (9) | 6* 7* (7) | ||
1250 | 0* 0* (0) | 0* 0* (0) | 12* 7* (10) | 0* 0* (0) | 0* 0* (0) | ||
5000 | 0* 0* (0) | 0* 0* (0) | 1* 4* (3) | 0* 0* (0) | 0* 0* (0) | ||
Positive controls | none | Name | AF-2 | NaN3 | AF-2 | AF-2 | ICR-191 |
Dose (µg/plate) | 0.01 | 0.5 | 0.01 | 0.1 | 1.0 | ||
Colony count / plate | 453 459 (456) | 395 407 (401) | 102 116 (109) | 357 387 (372) | 2412 2172 (2292) | ||
none | Name | 2AA | 2AA | 2AA | 2AA | 2AA | |
Dose (µg/plate) | 1.0 | 2.0 | 10.0 | 0.5 | 2.0 | ||
Colony count / plate | 832 823 (828) | 252 254 (253) | 436 453 (445) | 272 385 (279) | 105 112 (109) | ||
Positive control substances: AF-2: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide; NaN3: sodium azide; ICR-191: 6-chloro-9-[3-(2-chloroethylamino)propylamino]-2-methoxyacridine dihydrochloride; 2AA: 2-aminoanthracene
*: concentrations inhibiting bacterial growth
Numbers in brackets indicate plate averages
Table 2: Test results of the main experiment
Metabolic activation Y/N | Dose of test substance (μg/plate) | Revertant colonies/plate | |||||
Base pair substitution | Frame shift | ||||||
TA 100 | TA 1535 | WP2 uvrA | TA 98 | TA 1537 | |||
S9 mix (–) | Negative control | 99 94 99 (97) | 7 8 10 (8) | 26 25 26 (26) | 14 24 14 (17) | 9 6 8 (8) | |
4.9 | 98 84 (91) | 7 10 (9) | 32 32 (32) | 22 20 (21) | 8 8 (8) | ||
9.8 | 107 114 (111) | 13 11 (12) | 33 29 (31) | 18 14 (16) | 7 7 (7) | ||
20 | 96 99 (98) | 5 6 (6) | 36 23 (30) | 21 19 (20) | 8 10 (9) | ||
39 | 109 106 (108) | 7 13 (10) | 26 32 (29) | 17 21 (19) | 7 5 (6) | ||
78 | 93 97 (95) | 9 10 (10) | 24 36 (30) | 18 14 (16) | 7 8 (8) | ||
156 | 98* 99* (99) | 7* 6* (7) | 25 28 (27) | 14 13 (14) | 4 8 (6) | ||
313 | 0* 0* (0) | 0* 0* (0) | 0* 0* (0) | 10* 0* (5) | 0* 0* (0) | ||
S9 mix (+) | Negative control | 113 125 134 (124) | 8 9 10 (9) | 32 30 35 (32) | 33 25 29 (29) | 13 15 19 (16) | |
4.9 |
| 8 11 (10) |
|
|
| ||
9.8 | 116 102 (109) | 7 9 (8) | 35 33 (34) | 29 29 (29) | 15 16 (16) | ||
20 | 109 118 (114) | 7 7 (7) | 26 25 (26) | 33 27 (30) | 19 17 (18) | ||
39 | 102 125 (114) | 8 10 (9) | 33 35 (34) | 26 30 (28) | 17 19 (18) | ||
78 | 111 111 (111) | 7 10 (9) | 37 30 (34) | 30 33 (32) | 17 16 (17) | ||
156 | 93 90 (92) | 8 7 (8) | 35 32 (34) | 29 24 (27) | 15 17 (16) | ||
313 | 74* 78* (76) | 0* 4* (2) | 21* 21* (21) | 21* 16* (19) | 0* 8* (4) | ||
Positive controls | none | Name | AF-2 | NaN3 | AF-2 | AF-2 | ICR-191 |
Dose (µg/plate) | 0.01 | 0.5 | 0.01 | 0.1 | 1.0 | ||
Colony count / plate | 438 429 (434) | 477 485 (481) | 99 108 (104) | 428 430 (429) | 1832 1852 (1842) | ||
none | Name | 2AA | 2AA | 2AA | 2AA | 2AA | |
Dose (µg/plate) | 1.0 | 2.0 | 10.0 | 0.5 | 2.0 | ||
Colony count / plate | 836 823 (830) | 290 303 (297) | 828 862 (845) | 338 342 (340) | 131 126 (129) | ||
Positive control substances: AF-2: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide; NaN3: sodium azide; ICR-191: 6-chloro-9-[3-(2-chloroethylamino)propylamino]-2-methoxyacridine dihydrochloride; 2AA: 2-aminoanthracene
*: concentrations inhibiting bacterial growth.
Numbers in brackets indicate plate averages
Table 3: Background data (Historical control data: September 7, 2012 to February 1, 2013)
TA100 | ||||
Strain | Neg. ctrl. (S9-) | Neg. ctrl. (S9+) | Pos. ctrl (S9-) AF-2 (0.01 µg/plate) | Pos. ctrl (S9+) 2AA (1.0 µg/plate) |
|
|
|
|
|
No. of data | 64 | 64 | 64 | 64 |
Average | 97 | 105 | 566 | 835 |
SD | 10.1 | 12.8 | 60.8 | 80.3 |
|
|
|
|
|
TA1535 | ||||
|
|
|
|
|
Strain | Neg. ctrl. (S9-) | Neg. ctrl. (S9+) | Pos. ctrl (S9-) NaN3 (0.5 µg/plate) | Pos. ctrl (S9+) 2AA (2.0 µg/plate) |
|
|
|
|
|
No. of data | 32 | 32 | 32 | 32 |
Average | 12 | 11 | 491 | 257 |
SD | 3.1 | 2.9 | 72.9 | 27.7 |
|
|
|
|
|
WP2 uvrA | ||||
|
|
|
|
|
Strain | Neg. ctrl. (S9-) | Neg. ctrl. (S9+) | Pos. ctrl (S9-) AF-2 (0.01 µg/plate) | Pos. ctrl (S9+) 2AA (10.0 µg/plate) |
|
|
|
|
|
No. of data | 32 | 32 | 32 | 32 |
Average | 26 | 28 | 117 | 685 |
SD | 5.3 | 6.0 | 15.4 | 101.7 |
|
|
|
|
|
TA98 | ||||
|
|
|
|
|
Strain | Neg. ctrl. (S9-) | Neg. ctrl. (S9+) | Pos. ctrl (S9-) AF-2 (0.1 µg/plate) | Pos. ctrl (S9+) 2AA (0.5 µg/plate) |
|
|
|
|
|
No. of data | 103 | 103 | 103 | 103 |
Average | 17 | 26 | 457 | 323 |
SD | 2.9 | 4.3 | 40.7 | 39.3 |
|
|
|
|
|
TA1537 | ||||
|
|
|
|
|
Strain | Neg. ctrl. (S9-) | Neg. ctrl. (S9+) | Pos. ctrl (S9-) ICR-191 (1.0 µg/plate) | Pos. ctrl (S9+) 2AA (2.0 µg/plate) |
|
|
|
|
|
No. of data | 33 | 33 | 33 | 33 |
Average | 7 | 19 | 2588 | 118 |
SD | 1.8 | 2.9 | 830.5 | 16.0 |
AF-2: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide; NaN3: sodium azide; ICR-191: 6-chloro-9-[3-(2-chloroethylamino)propylamino]-2-methoxyacridine dihydrochloride; 2AA: 2-aminoanthracene
Applicant's summary and conclusion
- Conclusions:
- From the results in this study and under these test conditions, 1-(2,2,4-trimethylquinolin-l (2H)-yl)ethanone was assessed not to be mutagenic.
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